F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.123181.3 Research Article Articles Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1

[version 3; peer review: 5 not approved]

 Tandio Saputro Shania Safera Investigation Methodology Project Administration Software Writing – Original Draft Preparation https://orcid.org/0000-0001-6512-9612 1 Wahyunita Khayu Investigation Methodology Validation Writing – Original Draft Preparation 2 Nurhasanah Astutiati Investigation Methodology Project Administration Supervision Validation Writing – Original Draft Preparation https://orcid.org/0000-0003-0912-6913 2 Nugraha Yudhi Conceptualization Formal Analysis Funding Acquisition Investigation Methodology Supervision Validation Visualization Writing – Original Draft Preparation https://orcid.org/0000-0003-1186-4093 3 4 Faizal Irvan Methodology Validation https://orcid.org/0000-0002-7555-0678 1 2 Pambudi Sabar Conceptualization Data Curation Formal Analysis Investigation Methodology https://orcid.org/0000-0003-0703-437X 2 Wibowo Syahputra Formal Analysis Visualization Writing – Original Draft Preparation Writing – Review & Editing 3 Pramono Andri Pramesyanti Conceptualization Formal Analysis Funding Acquisition Investigation Methodology Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-6516-9814 a 3 5 6 Department of Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Tangerang, Jakarta Selatan, 15345, Indonesia Centre for Vaccine and Drug Research, National Research and Innovation Agency Republic of Indonesia, Kawasan Puspiptek Serpong, Serpong, 15314, Indonesia Research Centre of Molecular Biology Eijkman, National Reseach and Innovation Agency Republic of Indonesia, Kawasan Sains Teknologi Soekarno, Cibinong, 16911, Indonesia National Reseach and Innovation Agency Republic of Indonesia, Jakarta, Jakarta Pusat, 10340, Indonesia Stem Cell and Tissue Engineering Research Center, Universitas Pembangunan Nasional Veteran Jakarta, Jakarta, Jakarta Selatan, 12450, Indonesia Medical Faculty, Universitas Pembangunan Nasioinal Veteran Jakarta, Jakarta, Jakarta Selatan, 12450, Indonesia andri.pramesti@upnvj.ac.id

No competing interests were disclosed.

 25 11 2024 2023 12 1 22 10 2024 Copyright: © 2024 Tandio Saputro SS et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Green Fluorescent Protein (EGFP) is widely used as a reporter gene, aiding in protein recovery and transduction studies. In this study, EGFP was tagged with eleven arginine residues (PolyR) and six histidine residues (His-tag) for purification. The aim was to enhance the synthesis of EGFP-PolyR in Saccharomyces cerevisiae and evaluate the effects of polyarginine modification on protein stability and expression levels. The expression of EGFP and EGFP-PolyR in S. cerevisiae was assessed through fluorescence measurements and protein levels. Structural analyses were conducted using in silico tools to investigate changes in beta strands and helices, which were validated through Western blots. Results showed that EGFP-PolyR maintained similar fluorescence levels to EGFP, but with notable structural changes. EGFP-PolyR's final beta strand terminates at Ala228, compared to Gly229 in EGFP, affecting the beta sheet's stability. Structural modifications also included altered helix lengths, with a longer helix 10 and shorter helix 9 in EGFP-PolyR. These alterations, along with shifts in helix-helix interactions, contribute to destabilization. Additionally, EGFP-PolyR exhibited unique gamma coils absent in EGFP, further differentiating its structure. The structural changes led to decreased protein expression and solubility, as indicated by Western blot analysis, with EGFP-PolyR showing significantly lower expression levels. The findings suggest that EGFP-PolyR is prone to aggregation and misfolding, characteristics often associated with aggregation-prone proteins.In conclusion, the polyarginine modification significantly impacts the structural integrity, stability, and solubility of EGFP. While fluorescence is retained, these changes hinder protein detectability and purification, highlighting the importance of considering structural alterations when modifying reporter proteins for experimental use.

 Saccharomyces cerevisiae INVSc1 protein expression EGFP protein protein transductions. The works were financially supported by the Universitas Pembangunan Nasional Veteran Jakarta, DRPM Grant from Kemendikbud. ContractNr.001/UN.61.4/2020 Nr.001/UN.61.4/2021 Nr.046/E5/PG.02.00.PT/2022 The works were financially supported by the Universitas Pembangunan Nasional Veteran Jakarta, (Contract Nr. 001/UN.61.4/2020 and Nr. 001/UN.61.4/2021) DRPM Grant from Kemendikbud. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 2

In this version, There are several changes and additions to the paper : 1.                  We have added Dr. Syahputra Wibowo as an author because he conducted the bioinformatics analysis for this revision.  2.                  There are several parts that we decided to change this paper, namely: -          We have changed the title of this manuscript to reflect the additional data we have included. -          Introduction part, we changed it according to the bioinformatics analysis results. -          In the methodology section, we have incorporated protein structure analysis and structural analysis, thus completing the bioinformatic analysis. -          In the results section, we have incorporated additional bioinformatic analysis images and comprehensive structural analysis tables for EGFP and EGFP-PolyR. -          In the discussion section, the text has been modified and augmented in accordance with the incorporation of bioinformatic analysis data. -          In conclusion, the conclusion was also modified in accordance with the alterations made to other sections of the manuscript. -          In the extended data section, the figure previously published on figshare has been updated, and a new link has been included in this paper. -          In the reference section, some references were deleted and new ones were added in accordance with the revisions made to this paper.

 Introduction

Enhanced green fluorescent protein (EGFP) has often been used as a reporter for protein transport, especially in eukaryotic cells such as mammalian and yeast cells. EGFP was derived from green fluorescent protein (GFP), a commonly used reporter protein that does not require a substrate for it to fluoresce. The EGFP gene is a mutated version of the GFP gene, with P64L and S65T mutations. These mutations cause a higher emission intensity in EGFP relative to that of the wild-type GFP, and better thermal stability of the EGFP protein, especially in eukaryotic cells. 1 EGFP can be used as a marker in optimizing protein isolation procedures and their functions. The fluorescence makes it easier for us to observe the localization of the expression of the protein. The intensity of EGFP fluorescence can also indicate the level of expression of the protein in a system. EGFP can also be used to study protein transfer, such as analysis of the work of a protein transduction system for cell reprogramming purposes.

One of the uses of cell reprogramming is manufacturing induced pluripotent stem cells. Induced pluripotent stem cells are expected to be used as therapeutic cells for patients. Cell reprogramming can be done using protein transduction, viral transduction, or chemicals. Cell reprogramming by viral transduction can activate oncogenic genes, whereas chemical methods are highly unspecific and cannot be interpreted clearly. 2 , 3 On the other hand, protein transduction is reputed as a harmless method to deliver protein precisely. However, protein transduction has its disadvantages, such as low transduction efficiency in host cells. Therefore, research is needed to increase transduction proteins by modifying the target protein.

Polyarginines are short polypeptides classified as non-amphipathic cell-penetrating peptides (NaCPP) and have cationic characteristics, including binding anionic lipid membranes; therefore, polyarginine can enter through the plasma membrane of eukaryotic cells. As a result, the NaCPP class is less toxic than the primary amphipathic and secondary cell-penetrating peptide (CPP). The NaCPP class uses endocytosis mechanisms with low concentrations for cellular uptake. 4 The amount of added arginine amino acid residues affect the cellular uptake’s efficiency level, whereas more arginine residues have a better efficiency level. Adding more than 15 amino acid residues decreases intracellular delivery efficiency. 5 Proteins fused to polyarginine are more efficiently transduced into human iPS cells compared to those without the addition of CPP. 6

In this research, we designed and constructed two proteins, EGFP and EGFP-PolyR. EGFP-PolyR is an EGFP protein with the addition of eleven arginine residues (polyarginine – polyR) at the C terminal of EGFP to be used as a transduction protein model for cell reprogramming purposes. The gene encoding EGFP and EGFP-PolyR was synthetically constructed and expressed in Saccharomyces cerevisiae INVSc1. The study aimed to investigate the pattern of EGFP-PolyR protein expression in S. cerevisiae INVSc1 compared to EGFP. This paper aims to contribute to the current understanding of structural modifications when optimizing reporter proteins for experimental applications.

 Methods Plasmid design and construction

The gene encoding EGFP (GenBank Accession #U55762) and EGFP- PolyR (was synthetically made by IDT Custom Gene Synthesis cloned in a plasmid called pEGFP- PolyR, in such a way that it allowed sub-cloning of the gene with and without the polyR sequence, by using a different combination of restriction enzymes. In addition to the 11× arginine residues (polyR), codons encoding 6× histidine residues (6×His-tag) were also added downstream to the polyR codons. The synthetic gene was sub-cloned into pYES2CT, a Saccharomyces cerevisiae expression plasmid, during which the polyarginine codon either remained fused to the EGFP gene or was removed, creating recombinant pYES2CT pYES2CT-EGFP-PolyR or pYES2CT- EGFP, respectively. Since the pYES2CT plasmid has its own 6×His-tag downstream to its Multiple Cloning Sites (MCS), positioned in-frame with the EGFP and EGFP-PolyR codons, the expressed EGFP was expected to have one 6×His-tag, whereas the EGFP-PolyR were expected to have two.

The intended digested fragments sized 821 bp (EGFP-PolyR) and 733 bp (EGFP), were gel-purified using GenepHlow Gel/PCR kit (DFH004) (GeneAid, Taiwan) with 20 μL elution buffer. The DNA concentration was measured using a BioDrop-Duo UV/Vis spectrophotometer (Thomas Scientific, United States). Then, the DNA was stored at -20°C. Ligation was carried out following the T4 DNA ligase procedure as described by the manufacturer (Promega, United States). The ligation was performed at 4°C overnight, with a plasmid-insert volume ratio of 1:10.

 Transformation of <italic toggle="yes">E. coli</italic> TOP10

The transformation into Escherichia coli TOP10 was carried out following the method of Abyadeh et al. (2017). 7 Recombinant E. coli cells were cultured in LB with 100 μg/mL ampicillin. Transformation by heat shock was carried out using the Scilogex SCI-100HCM-Pro Digital Thermal Mixer (United States) at 42°C for 90 seconds. The transformants were selected on LB agar with100 mg/mL ampicillin (LB Agar+Amp). Colony PCR was performed on selected transformants with pYES2CT-specific primers (Forward: 5′-AATATACCTCTATACTTTAACGTC-3′; Reverse 5′-GAGGGCGTGAATGTAAGCGTGAC-3′) to verify the presence of intended inserts. PCR was done using Biometra Tone Thermal Cyclers (Analytik Jena AG, Germany) and carried out following the procedure from MyTaq Red Mix (Bioline, UK) manufacturer, for 35 cycles, with predenaturation (95°C) for 1 minute, denaturation for 15 seconds, annealing (55°C) for 15 seconds, elongation (72°C) for 10 seconds, and post elongation for 5 minutes.

 Transformation of <italic toggle="yes">Saccharomyces cerevisiae</italic> INVSc1

pYES2CT-EGFP- PolyR and pYES2CT-EGFP plasmids were isolated using ‘WizardPlus SV minipreps DNA purification kit (A1330) (Promega, United States)’ after being propagated in E. coli TOP10. Isolated plasmids were used to transform Saccharomyces cerevisiae INVSc1 with the lithium acetate (LiAc) method. 8 Transformants were selected on minimal medium (MM) agar without uracil. Plasmids were isolated from transformant colonies using the MP Biomedicals yeast plasmid isolation kit (MP Biomedicals, United States, Cat. 112069400). PCR was carried out to confirm the success of the transformation using Biometra Tone Thermal Cyclers (Analytik Jena AG, Germany) and carried out following the procedure from MyTaq Red Mix (Bioline, UK) manufacturer, for 35 cycles, with an annealing temperature of 55°C. Colony PCR was performed on selected transformants with pYES2CT-specific primers (Forward: 5′-AATATACCTCTATACTTTAACGTC-3′; Reverse 5′-GAGGGCGTGAATGTAAGCGTGAC-3′), to verify the presence of intended inserts. PCR was done using Biometra Tone Thermal Cyclers (Analytik Jena AG, Germany) and carried out following the procedure from MyTaq Red Mix (Bioline, UK) manufacturer, for 35 cycles, with predenaturation (95°C) for 1 minute, denaturation for 15 seconds, annealing (55°C) for 15 seconds, elongation (72°C) for 10 seconds, and post elongation for 5 minutes.

 Expression of the modified protein in <italic toggle="yes">Saccharomyces cerevisiae</italic>

S. cerevisiae’s fast-growing diploid strain INVSc1 (Invitrogen Corporation) was used as a host cell for protein expression. The yeasts were cultured in yeast-extract peptone dextrose (YEPD) medium. Recombinant cells of S. cerevisiae INVSc1 were cultured in a minimal medium (MM) without uracil. Protein expression followed the user guide of the pYES2CT plasmid (Invitrogen, 2003), with several modifications. A single colony of recombinant S. cerevisiae INVSc1 carrying pYES2CT-EGFP or pYES2CT-EGFP- PolyR was inoculated into 3 mL of MM medium; the culture was then incubated at 30°C 230 rpm for 48 hours. After that, a 1-mL culture was taken to measure its OD600 value. Culture with OD600 = 0.4 were subcultured into 100 mL of induction medium (MM medium containing 2% galactose and 1% raffinose). The cultures were further incubated after adding the induction medium for 48 to 72 hours. The culture was centrifuged at 5000 rpm for 10 minutes to harvest and stored in a freezer at -80 °C until further use.

 Fluorescent microscope visualization of EGFP and EGFP-PolyR Expression

A 1 mL 10× diluted culture was centrifuged at 5000 rpm for five minutes. The pellet was then fixed with 500 μL 3.7% formaldehyde for 30 minutes. The cell suspension was centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. as the remaining pellet was resuspended in 1 mL PBS 1×. A 50-μL fixed-cell suspension was placed in a well of 96-well plates (Thermo Scientific) to visualize the EGFP fluorescence under an inverted fluorescent microscope (Zeiss Axiovert 40 CFL, UK), (40× magnification, λ ex 488 nm and λ em 507 nm). Fluorescent intensity was measured using ImageJ software, and delta intensity was calculated as follows: ∆ Intensity t = Intensity t − Intensity 0 where,

Intensity t is the fluorescent intensity at time t post-inoculation to induction medium

Intensity 0 is the fluorescent intensity at time 0 post-inoculation to induction medium

 Statistical analysis

All data were expressed as mean ± standard deviation. All statistical analyses were performed using SPSS software (IBM SPSS Statistics 24). The differences between groups were analyzed using one-way analysis of variance (ANOVA) after passed the homogeneity test and normality test, then proceeded to the Bonferroni post-hoc test. A p-value < 0.05 was considered as statistically significant.

 Detection of expressed protein

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out following the modified Iqbal and Ahmad (2016) method. The protein sample was mixed with 6× SDS gel loading buffer (600 mM Tris-Cl pH 6.8, 12% SDS, 30% glycerol (v/v), 0.03% bromophenol blue, and 20 mM DTT) and ran on 10 % SDS-PAGE gel at 100 V for 110 minutes until the bromophenol blue dye reached approximately 0.5 cm from the lower limit of the gel. 9

Western blotting was also carried out following the method of Iqbal and Ahmad (2016), with several modifications. Protein was transferred from gel to membrane (transfer buffer: 300 mM glycine, 300 mM Tris, 0.05% SDS) using Pierce Power Blotter (Thermo Scientific, United States) set at 25 kV, 1.3 A for 25 minutes. Then, the membrane was washed six times with PBST (PBS, 1% Tween-20) before applying the primary antibody (Anti-6×His-Tag monoclonal antibody (Invitrogen, catalog number MA1-21315) in blocking buffer (5% skim milk (Merck, Germany) in PBST), with 1:3000 ratio). Then the membrane was incubated at 4°C overnight before applying the secondary antibody (Goat anti-mouse IgG HRP, Invitrogen, catalog number 31430 in blocking buffer, with a ratio of 1:20,000 ratio) for 60 minutes with shaking (70 rpm). Then, the substrate (SuperSignal West Femto Luminol, Thermo Scientific, United States) was used. The duration of the exposure was 20 minutes.

 Protein structure modeling

Protein structures were predicted using AlphaFold3, 10 a deep learning-based tool for accurate protein structure prediction. The input sequence for each protein was provided to AlphaFold3, which was run using the default settings. The predictions were performed on a high-performance computing cluster to ensure computational efficiency and accuracy. The output from AlphaFold2 included a predicted structure with confidence scores, which were used to assess the reliability of the model.

 Structural analysis

Ramachandran Plot Analysis: The stereochemical quality of the AlphaFol3-predicted structures was assessed using the Ramachandran plot, which was generated using PROCHECK. PROCHECK is a widely used tool for evaluating the geometry of protein structures, specifically analyzing the distribution of phi (φ) and psi (ψ) dihedral angles. The structural models generated by AlphaFold2 were analyzed using PROMOTIF, a tool available through the PDBSum web server. 11 PROMOTIF was employed to examine key structural features and motifs within the predicted protein structures. This included the identification of secondary structure elements, analysis of protein-protein and protein-ligand interfaces, and evaluation of the overall geometry of the model.

 Results Construction of Plasmid pYES2CT-EGFP and pYES2CT-EGFP-PolyR and Transformation into <italic toggle="yes">Saccharomyces cerevisiae</italic> INVSc1

After digests and ligation processes were successfully performed ( Figure 1 and Figure 2A), the ligation products, hereby called pYES2CT-EGFP and pYES2CT-EGFP-PolyR, for plasmid carrying egfp and egfp-poly R respectively, were propagated in E. coli TOP10. Plasmids isolated from this E.coli TOP10 that had been confirmed to have the correct size ( Figure 2B) and carried the genes ( Figure 2C) were then used to transform S. cerevisiae INVSc1. Screening of S. cerevisiae INVSc1 transformants was performed by PCR of purified plasmids. PCR results of positive colonies are presented in Figures 2D and 3.

The cloning process of genes encoding for EGFP and EGFP-PolyR into pYES2CT plasmid.

Insertion of EGFP-PolyR gene was performed using the enzymes BamH1 (position 519) and Not1 (position 577) (A), whereas EGFP gene was inserted into pYES2CT plasmid using BamHI and EcoRI, thus leaving the PolyR fragment out (B).

 pEGFP-Poly R (lane 3) and pYES2CT (lane 9) were cut with restriction enzymes <italic toggle="yes">Not</italic>I and <italic toggle="yes">Bam</italic>HI, where pEGFP-Poly R produced <italic toggle="yes">egfp-polyR </italic> gene (822 bp band), and pYES2CT became linearized with size 5932 bp.

pEGFP-Poly R (lane 5) and pYES2CT (lane 7) were cut with restriction enzymes EcoRI and BamHI, where pEGFP-Poly R produced Egfp gene (733 bp band), and pYES2CT produced vector (5905 bp band) (A). The 822 and 733 bp DNA were isolated from gel, and ligated to the 5932 and 5905 bp bands, respectively. Single digest ( BamHI) of plasmids isolated from E.coli TOP10 transformed with pYES2CT-EGFP (lane 3 and 4) and pYES2CT-EGFP-Poly R (lane 5 and 6), in comparison to wild type pYES2CT (lane 2), showed that the plasmid sizes were correct (B). Also, colony PCR of E.coli confirmed the presence of the intended genes as 1037bp and 1098bp bands, representing the Egfp and Egfp-polyR , respectively (C). Transformed yeast colony screening was performed by PCR of plasmids purified from the yeast, using pYES2CT-specific primers. As a control, pYES2CT plasmid was used (lane K). Lanes 1-15 showed PCR amplification results of plasmids isolated from S. cerevisiae culture, transformed with pYES2CT (lanes 1-5), pYES2CT-EGFP-Poly R (lanes 6-10), and pYES2CT-EGFP (lanes 11-15). Amplification of pYES2CT without insert (lane 1-5) shows the same size amplicons as a positive control (K), which are 334 bp, amplification of pYES2CT-EGFP-Poly R produced amplicons of 1097 bp (lane 6-10), while the pYES2CT-EGFP produced amplicons of 1036 bp (lane 11-15) (D). Lane M is GeneRuler 1kb DNA Ladder (Thermo Scientific SM0312).

 Positive <italic toggle="yes">E. coli</italic> TOP10 and <italic toggle="yes">S. cer</italic>evisiae INVSc1 transformants are carrying plasmids pYES2CT-EGFP (A3 and B3, respectively) and pYES2CT-EGFP-PolyR (A4 and B4, respectively).

Transformation positive controls are shown in A1 and B1, whereas the negative controls are in A2 and B2.

 The role of EGFP and EGFP-PolyR addition on the growth curve of <italic toggle="yes">Saccharomyces cerevisiae</italic> INVSc1

The growth curve showed that post-induction S. cerevisiae INVSc1 stayed longer at the exponential phase when not carrying any plasmid in comparison to the same strain with pYES2CT plasmids ( Figure 4). The plasmid-carrying yeasts seemed to reach stationary phase 24 hours after inoculation to induction medium ( Figure 4B and C), whereas the wild-type culture stayed at exponential phase for up to 64 hours ( Figure 4A) before slowing down its growth. There is a significant decrease of the growth curve in S. cerevisiae INVSc1 carrying the plasmids EGFP and EGFP-PolyR, compared to the wildtype. The significant decrease was seen from 48 – 72 hours ( Figure 4D).

Growth curve comparison of <italic toggle="yes">Saccharomyces cerevisiae</italic> INVSc1 without plasmid (A), carrying pYES2CT- EGFP (B), carrying pYES2CT-EGFP-PolyR plasmid (C), in the induction medium (containing 2% galactose and 1% raffinose). The growth comparison of Saccharomyces cerevisiae INVSc1 on 48 hours and 72 hours (D).

Optical densities were measured at λ=660nm, 48 hours, and 72 hours post-inoculation to induction medium of the wild-type and recombinant S. cerevisiae (D). The result reported are mean of four replicates while the error bar indicates its standard deviation. Mean data accompanied by different letter in each time point are significantly different (Bonferroni test, p < 0.05).

The fluorescence produced post-inoculation to induction medium by each recombinant yeast and wild-type yeast is presented in Figure 5A. The Δintensity was calculated and presented as a graph in Figure 5B. There was a significant difference between wildtype fluresens expression with recombinant yeast carrying pYES2CT EGFP and pYES2CT EGFP PolyR-plasmid on 48 hours and 72 hours. The addition of pYESC2 EGFP and pYES2CT EGFP PolyR plasmids significantly inhibited the proliferation of S. cerevisiae starting at 48 hours.

Western blotting using anti-his-tag mouse monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) proved that the EGFP-PolyR and EGFP proteins were expressed in S. cerevisiae INVSc1 at similar ( Figure 6).

The fluorescence expressed by <italic toggle="yes">S. cerevisiae</italic> INVSc1 carrying no plasmid (wild-type), and carrying pYES2CT-EGFP and pEGFP-PolyR plasmids at 48, and 72 hours post-inoculation to induction medium (A), the calculated Δintensity of each yeast at 48- and 72-hour post inoculation to induction medium (B).

The result reported are mean of six replicates while the error bar indicates its standard deviation. Mean data accompanied by different letter in each time point are significantly different (Bonferroni test, p < 0.05).

 Western blotting of protein expressed at 48 hours post-inoculation to induction medium by <italic toggle="yes">S. cerevisiae</italic> INVSc1 carrying no plasmid (Lane 1 and 2), pYES2CT-EGFP (Lane 3 and 4), and pYES2CT-EGFP-PolyR (Lanes 5 and 6).

The Western blot was performed using an anti-his-tag antibody as the primary antibody.

 Bioinformatic analysis

The beta strand structures of EGFP-Poly R and EGFP exhibit a critical difference in the length of the final beta strand. In EGFP-Poly R, the last beta strand ends at Ala228, whereas in EGFP, it extends one residue further to Gly229. This difference could impact the structural stability of the beta sheet, as the absence of the glycine residue in EGFP-Poly R might reduce the flexibility and stability of this structural element. In addition to differences in beta strands, there are notable variations in the helical structures of EGFP and EGFP-Poly R. EGFP features a total of 11 helices, including both G and H types, while EGFP-Poly R has only 10 helices, missing one compared to EGFP ( Table 1 and Table 2). This reduction in helices could significantly affect the overall stability and integrity of the protein structure, as helices play a crucial role in maintaining protein stability. Notably, helix 10 in EGFP spans from Leu251 to Ser253, comprising 3 residues, whereas in EGFP-Poly R, it extends to 5 residues, from Pro265 to Leu269 ( Figure 7 and Figure 8). This extension may reflect a structural adjustment due to the additional arginine residues in the Poly R sequence. Conversely, helix 9 in EGFP-Poly R, spanning from Pro245 to Trp248, shows decreased length and stability with a lower pitch and rise per residue compared to EGFP, suggesting potential instability. Both EGFP and EGFP-Poly R deviate from ideal helical parameters, but EGFP-Poly R exhibits slightly greater deviations in helix 9, indicating possible structural strain from the Poly R modification. The extension of helix 10 by two additional residues in EGFP-Poly R might be a direct consequence of this modification, altering the local structural environment. These differences in the beta strands and helices between EGFP and EGFP-Poly R likely contribute to the overall destabilization of the protein. The shortening of the final beta strand, combined with changes in helix lengths and stability, particularly the extension of helix 10 could lead to misfolding or reduced structural integrity of EGFP-Poly R ( Figure 8). Additionally, the interactions between helices show some important differences. The helix-helix interactions between EGFP and EGFP-Poly R reveal subtle yet significant differences that could impact the stability of the protein. Both proteins share similarities in certain interactions, such as those between helices A1 and A5, A1 and A6, A2 and A3, and A5 and A6, with nearly identical distances and angles. However, differences arise in the interactions between helices A5 and A8, and A8 and A9. In EGFP, the A5-A8 interaction has a distance of 10.6 Å with an angle of -102.9°, while in EGFP-Poly R, this distance shortens to 10.0 Å with an angle of -100.5°. More notably, the A8-A9 interaction shows a distance of 2.6 Å with an angle of -79.3° in EGFP, but in EGFP-Poly R, the distance increases to 3.2 Å with a significant angle shift to -94.9°, and an increase in the number of interacting residues from 4 to 6. These differences suggest altered packing and potential destabilization of the protein structure in EGFP-Poly R. The changes in helix-helix interaction, particularly between A8 and A9, could contribute to a less stable conformation in EGFP-Poly R.

Comprehensive structural of EGFP.
Structure type Start End Sequence Turn Type/Type Length/No. Resid Class/ H-Bond/Topology/Interaction Type/Helices
Beta Hairpins
Val12 Val23 12 Strands 2:2 IP
His26 Asp37 12 Strands 4:4 I
Tyr93 Phe101 9 Strands 3:5
Asn106 Glu116 11 Strands 2:2 IIP
His149 Asp156 8 Strands 4:4 I
Gly161 Asn171 11 Strands 3:5
His200 Ser209 10 Strands 10:10
Beta Bulges
Phe101 Asp104 Gly105 Classic Antiparallel H-Bond: Yes
Ile172 Gly175 Ser176 G1 Antiparallel H-Bond: Yes
Strands
Val12 Val23 12 Residues
His26 Asp37 12 Residues
Lys42 Cys49 8 Residues
Tyr93 Phe101 9 Residues
Asn106 Glu116 11 Residues
Thr119 Ile129 11 Residues
His149 Asp156 8 Residues
Gly161 Asn171 11 Residues
Val177 Pro188 12 Residues
His200 Ser209 10 Residues
His218 Gly229 12 Residues
Helices
Lys4 Phe9 Helix G 6 Residues
Trp58 Leu61 Helix G 4 Residues
Val62 Leu65 Helix G 4 Residues
Gln70 Phe72 Helix G 3 Residues
Asp77 His82 Helix G 6 Residues
Phe84 Ser87 Helix H 4 Residues
Lys157 Lys159 Helix H 3 Residues
Met234 Arg243 Helix H 10 Residues
Pro245 Trp248 Helix H 4 Residues
Leu251 Ser253 Helix G 3 Residues
Pro265 Gly268 Helix G 4 Residues
Helix-Helix Interactions A1 A5 G G n c 1 interacting residue
A1 A6 G H c 3 1 interacting residue
A2 A3 G H c n 4 interacting residues
A5 A6 G H c n 1 interacting residue
A8 A9 G H I 2 interacting residues
Beta Turns
Asp22 Gly25 DVNG IV H-Bond: Yes
Val23 His26 VNGH I H-Bond: Yes
Asp37 Tyr40 DATY I H-Bond: Yes
Ala38 Gly41 ATYG I
Cys49 Gly52 CTTG I H-Bond: Yes
Leu65 Gly68 LTYG I
Thr66 Val69 TYGV I H-Bond: Yes
Tyr67 Gln70 YGVQ IV
Ala88 Glu91 AMPE VIa1
Met89 Gly92 MPEG I
Phe101 Asp104 FKDD IV H-Bond: Yes
Phe115 Asp118 FEGD IV
Glu116 Thr119 EGDT II' H-Bond: Yes
Lys132 Gly135 KEDG I H-Bond: Yes
Gly135 Leu138 GNIL IV
Asn136 Gly139 NILG I
Ile137 His140 ILGH I H-Bond: Yes
Ile172 Gly175 IEDG I H-Bond: Yes
Asp211 Glu214 DPNE I H-Bond: Yes
Gly229 Leu232 GITL IV H-Bond: Yes
Gamma Turns
His82 Phe84 HDF INVERSE
Glu143 Asn145 EYN INVERSE
Gly268 Asp270 GLD INVERSE
Thr272 Thr274 TRT INVERSE
Gly275 His277 GHH INVERSE
His276 His278 HHH INVERSE
His278 His280 HHH INVERSE
Comprehensive Structural of EGFP-POLY R.
Structure type Start End Sequence Type/Turn Type Length/No. Resid Class/Interaction/H-Bond/Topology
Beta Sheet Mixed 11 strands Topology: -1 -1 5X -1-1 5-1 -1 3 1
Beta Hairpins
Val12 Val23 Hairpin 12 residues 2:2 IP
His26 Asp37 Hairpin 12 residues 4:4 I
Tyr93 Phe101 Hairpin 9 residues 3:5
Asn106 Glu116 Hairpin 11 residues 2:2 IIP
His149 Asp156 Hairpin 8 residues 4:4 I
Gly161 Asn171 Hairpin 11 residues 3:5
His200 Ser209 Hairpin 10 residues 10:10
Beta Bulges
Phe101 Asp104 Gly105 Bulge Antiparallel H-Bond: Yes
Ile172 Gly175 Ser176 Bulge Antiparallel H-Bond: Yes
Strands
Val12 Val23 Strand 12 residues
His26 Asp37 Strand 12 residues
Lys42 Cys49 Strand 8 residues
Tyr93 Phe101 Strand 9 residues
Asn106 Glu116 Strand 11 residues
Thr119 Ile129 Strand 11 residues
His149 Asp156 Strand 8 residues
Gly161 Asn171 Strand 11 residues
Val177 Pro188 Strand 12 residues
His200 Ser209 Strand 10 residues
His218 Ala228 Strand 11 residues
Helices
Lys4 Phe9 Helix 6 residues
Trp58 Leu61 Helix 4 residues
Val62 Leu65 Helix 4 residues
Gln70 Phe72 Helix 3 residues
Asp77 His82 Helix 6 residues
Phe84 Ser87 Helix 4 residues
Lys157 Lys159 Helix 3 residues
Met234 Arg243 Helix 10 residues
Pro245 Trp248 Helix 4 residues
Pro265 Leu269 Helix 5 residues
Helix-Helix Interactions A1 A5 G G n c 1 interacting residue
A1 A6 G H I c 3 interacting residues
A2 A3 G H c n 4 interacting residues
A5 A6 G H c n 1 interacting residue
A8 A9 G H I 2 interacting residues
Beta Turns
Asp22 Gly25 DVNG Turn IV H-Bond: Yes
Val23 His26 VNGH Turn I H-Bond: Yes
Asp37 Tyr40 DATY Turn I H-Bond: Yes
Ala38 Gly41 ATYG Turn I
Cys49 Gly52 CTTG Turn I H-Bond: Yes
Leu65 Gly68 LTYG Turn IV H-Bond: Yes
Thr66 Val69 TYGV Turn I H-Bond: Yes
Tyr67 Gln70 YGVQ Turn IV
Ala88 Glu91 AMPE Turn VIa1
Met89 Gly92 MPEG Turn I
Phe101 Asp104 FKDD Turn II H-Bond: Yes
Phe115 Asp118 FEGD Turn IV
Glu116 Thr119 EGDT Turn II H-Bond: Yes
Lys132 Gly135 KEDG Turn I H-Bond: Yes
Gly135 Leu138 GNIL Turn IV
Asn136 Gly139 NILG Turn I
Ile137 His140 ILGH Turn I H-Bond: Yes
Ile172 Gly175 IEDG Turn I H-Bond: Yes
Asp211 Glu214 DPNE Turn I H-Bond: Yes
Gamma Turns
His82 Phe84 HDF Turn INVERSE
Glu143 Asn145 EYN Turn INVERSE
Phe257 Gly259 FEG Turn INVERSE
Leu269 Ser271 LDS Turn INVERSE
Ser272 Arg275 TRT Turn INVERSE
Thr277 Arg278 GRR Turn INVERSE
Arg276 Gly277 TGR Turn INVERSE
Bioinformatic analysis of EGFP (yellow = 6x HisTag). Bioinformatic analysis of EGFP-Poly R (red = Poly R and yellow = 6x HisTag).

The comparison of beta turns between EGFP and EGFP-Poly R reveals several unique structural differences that highlight how modifications in the sequence can influence the protein's conformation. One notable difference is observed in the Leu65-Gly68 turn. EGFP exhibits a phi angle of -89.7° and a psi angle of -39.1°, while EGFP-Poly R shows a significantly more negative phi angle of -102.1° and a psi angle of -35.1°. This change in the phi angle suggests a more pronounced deviation in the turn's geometry in EGFP-Poly R compared to EGFP. Additionally, the chi1 angles are 51.1° for EGFP and 47.1° for EGFP-Poly R, reflecting an adjustment in the side-chain orientation due to the extended polyarginine sequence. Another distinct difference is found in the Phe115-Asp118 turn. EGFP has a phi angle of -125.8° and a psi angle of 100.9°, whereas EGFP-Poly R shows a less negative phi angle of -121.3° and a psi angle of 98.3°. The chi1 angles for EGFP are -65.5°, while for EGFP-Poly R, they are -66.0°. The shift in the phi and psi angles in EGFP-Poly R indicates a possible alteration in the turn’s local conformation, potentially affecting interactions with neighboring regions. The Asp211-Glu214 turn presents another unique aspect. EGFP has a phi angle of -63.7° and a psi angle of -16.3°, whereas EGFP-Poly R shows slightly different angles with a phi of -64.6° and a psi of -16.1°. This minimal difference in phi and psi angles, along with chi1 angles of 26.5° for EGFP and 26.8° for EGFP-Poly R, highlights subtle adjustments in the beta turn’s configuration. Another differences in the Leu65-Gly68 turn, in EGFP, this turn is of the I type, suggesting a more regular and stable loop structure at this position. In contrast, in EGFP-Poly R, the same region forms an IV type turn, indicating increased flexibility or a less well-defined structure, which could lead to differences in the local stability of the protein. The I type turn is a common secondary structure element in proteins where four consecutive residues create a turn in the polypeptide chain. In this type of turn, the first and fourth residues are hydrogen-bonded, creating a tight loop. The phi (Φ) and psi (Ψ) angles typically found in I type turns are around -60° for Φ and -30° to -60° for Ψ. These angles contribute to the formation of a stable turn, often found in beta-hairpins or at the end of beta strands. The IV type turn is a more flexible and less common turn compared to type I. It doesn’t conform to a specific set of phi and psi angles and is often seen as a more irregular or less tightly constrained turn. This flexibility means that IV type turns can vary more in their structure, leading to different folding patterns or conformations in the protein. Because of its variability, an IV type turn can introduce local flexibility or disorder in a protein structure, potentially affecting the overall stability of the region. Flexible or irregular turn like the IV type could make the protein more prone to misfolding or less resistant to environmental stressors. 12

The last one is the difference of gamma turns between EGFP and EGFP-Poly R reveals significant structural differences that likely influence their stability and functionality. EGFP features seven gamma turns ( Table 1), whereas EGFP-Poly R contains nine, indicating that the poly-arginine modification introduces additional structural elements ( Table 2). Among the common gamma turns, the His82 to Phe84 turn shows minor differences. In EGFP, the phi angle is -85.6°, psi is 82.4°, chi1 is -168.5°, with a CA distance of 6.0 Å. For EGFP-Poly R, the phi angle is -86.3°, psi is 83.0°, chi1 is -169.3°, and the CA distance remains 6.0 Å. Similarly, the Glu143 to Asn145 turn in EGFP has a phi angle of -84.5°, psi of 65.2°, chi1 of -171.6°, with a CA distance of 5.6 Å. In EGFP-Poly R, the corresponding values are phi -83.9°, psi 65.6°, chi1-170.0°, and CA distance 5.6 Å. EGFP also has unique gamma turns not present in EGFP-Poly R. The Gly268 to Asp270 turn, with a phi angle of -84.6°, psi of 69.5°, chi1 of -70.8°, and a CA distance of 5.8 Å, is missing in EGFP-Poly R. Its absence may lead to reduced flexibility and altered local folding. Similarly, the Thr272 to Thr274 turn, with phi of -72.7°, psi of 62.5°, chi1 of -78.9°, and CA distance of 5.4 Å, is also not found in EGFP-Poly R. The Gly275 to His277 turn, with phi of -65.9°, psi of 95.2°, chi1 of -86.4°, and a CA distance of 5.8 Å, is absent in EGFP-Poly R. On the other hand, EGFP-Poly R includes several unique gamma turns not observed in EGFP. The Phe257 to Gly259 turn, with phi of -61.9°, psi of 76.0°, chi1 of -74.2°, and a CA distance of 5.0 Å, introduces new conformational possibilities. The Leu269 to Ser271 turn, with phi of -75.5°, psi of 58.9°, chi1 of -152.5°, and a CA distance of 5.5 Å. The Thr272 to Thr274 turn in EGFP-Poly R, with phi of -56.1°, psi of 83.6°, chi1 of -80.3°, and a CA distance of 5.7 Å, differs from its counterpart in EGFP. Then Gly275 to Arg277 turn, with phi of -82.1°, psi of 91.4°, chi1 of -94.9°, and a CA distance of 6.0 Å, reflects a structural modification that might influence the protein's overall folding.

 Discussion The addition of PolyR residues did not affect the expression of EGFP fluorescence in the Saccharomyces cerevisiae cell

In this study, PolyR was added to EGFP protein to function as a cell-penetrating peptide (CPP), which is commonly used as a carrier of protein into cells. Arginine-rich CPP has been used as a carrier for the intracellular delivery of various bioactive molecules such as proteins, peptides, and nucleic acids. The number of arginine residues used as CPP can affect the efficiency of cellular uptake and cytosolic release. It is well understood that high cellular uptake efficiency is achieved with 8-12 residues. 13 Polyarginine as CPP also plays an essential role in forming hydrogen bonds with cell membrane protein groups. 14

The expression of EGFP protein, as inferred from the fluorescence and Western blot data, indicated that the induction of the recombinant cultures was successful. Analysis performed at 48 and 72 hours post-inoculation to induction medium showed no increase in fluorescent intensity with time ( Figure 5B). Thus, in the future, this could be used as a reference supporting that it would only take 48 hours of culturing for expression, hence being more time-efficient.

It is known that the addition of a CPP sequence can have a negative influence on recombinant protein yield. 15 It was reported that ten arginines (R10) residues were added to the superfolder GFP (sfGFP) N-terminal in pBAD plasmid containing arabinose-inducible sfGFP gene with a C-terminal His tag. No recombinant R10-sfGFP was expressed by E. coli TOP10. However, the unmodified sfGFP gene expressed over 30 mg/L protein under the same condition. 16 It was also reported that although the fluorescent protein obtained after purification showed a single band on SDS-PAGE, mass spectrometry (MS) analysis revealed truncation of the R10, leaving only two arginine residues present at the C-terminus upon expression when the position of R10 and His-tag was swapped relative to the sfGFP gene ( i.e. His tag at the N-terminus and R10 at the C-terminus). 17

In contrast to these previous findings, our result indicated that the addition of a PolyR group to the C-terminal of EGFP carrying C-terminal 6×His-tag did not seem to interfere with protein expression in S. cerevisiae INVSc1, as shown by the similar fluorescence ( Figure 5B) and protein ( Figure 6) levels expressed by yeasts carrying pYES2CT-EGFP band pYES2CT-EGFP-PolyR. In combination with the data from the proteins tertiary structural predictions and alignment, this expression result indicated that the conformational change caused by the addition of PolyR did not affect the protein expression.

It would be interesting to observe whether relocating the 6×His-tag or the PolyR to different terminals would improve the protein expression as well as provide ease of purification since it is feared that the PolyR can interfere with the binding of 6×His-tag to Ni-NTA resin to be used for purification or the anti-His-tag antibody used for detection of expression.

 Modified protein plasmid insertion causes a decrease in the growth curve of <italic toggle="yes">S. cerevisiae</italic>

The lower growth curve of the transformant S. cerevisiae compared to the wild-type can be due to the growth inhibition factors of S. cerevisiae activated due to the addition of MM induction medium (2% galactose and 1% raffinose). Adding raffinose to the growth medium of S. cerevisiae can activate the programmed cell death formation of acetic acid. Guaragnella's study (2013) suggests that S. cerevisiae is resistant to programmed cell death via acetic acid because S. cerevisiae can compensate by activating retrograde mitochondria. Although programmed cell death is activated, they are not necessarily inhibited by growth. 16 In addition, previous studies have shown that high-level expression of a EGFP protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes and growth defects. 17 , 18 This causes a significant decrease in the growth curve of yeast containing plasmids EGFP and EGFP PolyR. Even so, the low growth curve of the transformant S. cerevisiae seems does not affect the expression of EGFP fluorescence proteins ( Figures 5A and 5B and 2). Previous research has shown that PolyR expression in cells can cause protein translation stalls. 19 Results in experiments showed that the addition of PolyR at the C terminal did not cause the EGFP protein translation stall, only a decrease of the growth curve significantly. This is possible because our plasmid vectors have been designed to express them in sufficient quantities within the cell.

 Structural alterations of EGFP-Poly R lead to reduced Western blot detection

The reduced detection of EGFP-Poly R in Western blot assays, compared to native EGFP, stems from significant structural alterations caused by the poly-arginine modifications. EGFP-Poly R exhibits a truncated final beta strand and a reduced number of helices, leading to decreased stability and increased susceptibility to misfolding and aggregation. Additionally, changes in the lengths and angles of helices, as well as the introduction of new gamma turns, contribute to structural strain and potential instability. 20 These modifications result in reduced solubility and altered epitope accessibility, making EGFP-Poly R more prone to proteolytic cleavage during sample preparation. Consequently, while EGFP-Poly R is expressed within cells, its aggregated and unstable forms are less detectable in Western blots, unlike the more stable and detectable native EGFP. 21 Histag addition can cause protein instability. It would seem prudent to suggest that some cases of histag may result in increased aggregation, disruption to protein folding, proteolytic changes of cleavage, and a combination of these factors, which could be 8X more significant than 6X. 22 – 24

Conclusions

It is imperative to stress that poly-arginine causes significant changes with regard to the structure of EGFP and affects the stability and solubility of the protein. However, it is rather reassuring that the fluorescence of EGFP-PolyR is preserved at similar levels. However, the changes in the secondary structure elements, such as beta-strands, helices, and turns, as well as the increase in protein instability, should not be disregarded as they do influence the detectability and overall usefulness of the antibody in protein immobilization and isolation/analysis. Which proves the fact that structural alteration should be taken into account when optimizing the reporter proteins for the experimental purposes.

 Data availability Underlying data

Figshare: Growth curve of Saccharomyces cerevisiae with and without plasmid insertion. A. Growth curve of Saccharomyces cerevisiae without plasmid, https://doi.org/10.6084/m9.figshare.19759018.v1. 25

Figshare: The fluorescence of Saccharomyces cerevisiae without transformation and with transformation (pYES2CT- EGFP/ EGFP plasmid, pYES2CT-EGFP- PolyR/EGFP Polyarginine plasmid) at OD 600 on 48 hours and 72 hours, https://doi.org/10.6084/m9.figshare.19762699.v2. 26

Extended data

Figshare: Figure 1-7, https://doi.org/10.6084/m9.figshare.25388371. 27

Figshare: Attachment, https://doi.org/10.6084/m9.figshare.19767589.v8. 28

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgments

We would like to thank Program SAME Direktorat Sumber Daya, DRPM Ditjen Diktiristek, Kemendikbud Tahun 2022, and LPPM UPNVJ for their support in this study.

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Biol [Internet]. 2015 Aug1 [cited 2024 Oct 2];33:161–168. 26497286 10.1016/j.sbi.2015.09.002 PMC4858314 Tokuriki N Stricher F Schymkowitz J : The stability effects of protein mutations appear to be universally distributed. J. Mol. Biol [Internet]. 2007 Jun 22[cited 2024 Sep 26];369(5):1318–1332. 17482644 10.1016/j.jmb.2007.03.069 Saputro SST Wahyunita K Nurhasanah A : Growth Curve Optical Density of Sacharomyces cerevisiae wild-type, EGFP vector insertion and EGFP PolyR insertion.csv. figshare.Dataset.2022. 10.6084/m9.figshare.19759018.v1 Saputro SST Wahyunita K Nurhasanah A : The fluorescence of Saccharomyces cerevisiae without transformation and with transformation (pYES2CT- EGFP/ EGFP plasmid, pYES2CT-EGFP- PolyR/EGFP Polyarginine plasmid) at OD 600 on 48 hours. figshare.Dataset.2022. 10.6084/m9.figshare.19762699.v2 Saputro SST Wahyunita K Nurhasanah A : Figure 1-7. figshare. Figure. 2024. 10.6084/m9.figshare.25388371 Saputro SST Wahyunita K Nurhasanah A : Attachment. figshare. Figure. 2022. 10.6084/m9.figshare.19767589.v8 10.5256/f1000research.172954.r360640 Reviewer response for version 3 Perrine-Walker Francine M 1 Referee The University of Sydney, Sydney, New South Wales, Australia

Competing interests: No competing interests were disclosed.

 19 2 2025 Copyright: © 2025 Perrine-Walker FM 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The manuscript by Tandio Saputro and colleagues titled ‘Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1’ investigated how the reporter, Green Fluorescent Protein (EGFP) could be structurally modified by tagging it with eleven arginine residues (PolyR) and monitor how the protein stability and expression levels were affected by this structural change compared to EGFP. In silico studies and western blots were also used to demonstrate how such modifications not only affected the reporter protein’s structure but also had a negative effect on protein detectability and purification.

 The paper would be of interest to many readers especially those who use protein fluorescence tags. However, the paper does not provide a strong argument as to why it is important to understand polyR in the context of protein expression in cells or perhaps protein expression at the cell membrane surface.

Here are some suggestions to improve the paper addressing Partly comments (shown above)

1. for keywords please replace Saccharomyces cerevisiae INVSc1 protein expression, EGFP protein as they are already in the title. Other keywords could be used to increase the chances of the paper being seen by readers.

2. for figure 2, in Lane M, GeneRuler 1kb DNA Ladder was used. It would be helpful to have the sizes of the bands. Please use this website as a guide: https://tinyurl.com/23b9rufk

3. for culturing the yeast and E. coli transformants, were any antibiotics used etc. please state in the legend or in the method section.

4. for figure 5A. Though it was mentioned that images were taken at x40 with Zeiss Axiovert 40 CFL. Was there any possibility to observe the cells at a higher magnification? Or use oil at X100 objective? Please add a scale bar or mention the total magnification used in the legend.  What camera or image software was used for this section. Please mention in the method section. Do you think that fixing the cells for 30 minutes could have affected the GFP signal observed? This paper could be useful https://doi.org/10.2144/000113872  .  Any live images taken at high magnification?

As the authors had both EGFP and EGFP-PolyR in E. coli, did you observe similar expression patterns in E. coli under the microscope etc?

5. these references could be useful to improve the discussion section and to provide some evidence as to why these studies could be useful

a. Fuchs SM.et.al; 2005 (Ref 1)

b. Fenton D.et.al; 2020 (Ref 2)

c. Le et al; 2022 (Ref 3)

For the discussion perhaps – the focus could be on yeast as a eukaryotic cell instead of a mammalian cell.

The authors could provide a model of what they think the PolyR is doing and how is affecting GFP signal in the yeast cell or at the cell membrane as a final figure for the discussion section.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Yeast, molecular biology, cell biology, plant science, plant-microbe interactions

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References : Polyarginine as a multifunctional fusion tag. Protein Sci .2005;14(6) : 10.1110/ps.051393805 1538-44 15930002 10.1110/ps.051393805 : Cupid, a cell permeable peptide derived from amoeba, capable of delivering GFP into a diverse range of species. Sci Rep .2020;10(1) : 10.1038/s41598-020-70532-x 13725 32792509 10.1038/s41598-020-70532-x : Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis. Biotechnol Rep (Amst) .2022;35: 10.1016/j.btre.2022.e00754 e00754 35911505 10.1016/j.btre.2022.e00754 10.5256/f1000research.172954.r343340 Reviewer response for version 3 Guyot Stéphane 1 Referee https://orcid.org/0000-0002-2316-234X UMR Procédés Alimentaires et Microbiologiques, L'Institut Agro Dijon, Dijon, Bourgogne-Franche-Comté, France

Competing interests: No competing interests were disclosed.

 14 12 2024 Copyright: © 2024 Guyot S 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The new comments are presented in bold and are based on the previous report and the new version of the paper. In light of the new version of the paper with tracked changes and no direct response to the comments (except a summary of the changes), I have numbered the lines to assist the authors in linking my comments to the paper's content. I have numbered the first line as the line with the introduction word, then the lines where they are numbered continuously. This method is not conducive to enabling the reviewer to understand why some comments were not (or partly) taken into account, as debate and challenges are integral to the scientific process.

The main aim of this paper is to propose a method for expressing a EGFP carrying C-terminal PolyR plus 6xHis-tag in the diploid Saccharomyces cerevisiae INVSc1 strain. Such an engineering approach is proposed to study and improve the transduction process that can be used for cell reprogramming to produce pluripotent stem cells. In general, the paper presents the different stages of yeast transformation by insertion of an appropriately modified plasmid, the effect of the presence of EGFP flanked or not by a PolyR sequence on yeast growth and a protein structure prediction of the EGFP tagged or not with PolyR and 6xHis tag. At this stage, there may be room for improvement in the paper. Some suggestions for enhancements are presented below for your consideration.

Major comment

1) My main concern is the functionality of the proteins expressed in Saccharomyces cerevisiae. It is also a question of looking beyond the main objective of this study, which is to optimize the expression of these proteins. Although it would be tedious to carry out an experimental approach involving the cultivation of animal or human cells, I think that in the context of this study it would be interesting to ask whether post-translational modifications made by yeast, which cannot be detected by Western blotting, alter the desired functionality of modified EGFP (PolyR with or without 6xHis-tag) without affecting its fluorescence properties? To validate the present study, it would be advisable to extract the modified EGFP protein, bring it into contact with a human cell, and monitor its internalization (protein transduction) by fluorescence microscopy.

New comment: the functionality of the tagged EGFP has yet to be evaluated, which serves to reinforce the interest in this study.

Introduction section

2) From my point of view, the introductory section could be improved by not focusing only on one application based on stem cells, since there are a wide range of applications related to the use of protein transduction in various fields such as therapeutic delivery (drug and gene therapy), molecular biology research, regenerative medicine (stem cell engineering and tissue repair), vaccine development, diagnostics, and neuroscience.

New Comment: This section has been improved. I don't understand why the 6xHis tag is not mentioned at the end of the section (lines 33-40). Please mention it as you did in the abstract.

Methods section

3) Centrifugation steps: Please indicate the relative centrifugal force (x g) rather than the revolutions per minute (in rpm) throughout the paper.

New comment: not done.

4) Fluorescent Microscopy: Please clarify why yeasts were fixed with formaldehyde and not observed under a living state.

Please indicate the version of the ImageJ software and the plugins utilized for the measurement of fluorescence intensity. As the distribution of fluorescence intensity is not uniform throughout the yeast, it is necessary to specify how the analysis was conducted. In particular, it is important to ascertain which pixels were taken into account. Please clarify whether the ratio of fluorescence intensity to yeast was taken into account. Please indicate the number of cells that were counted. The optimal approach is to conduct an analysis at the single-cell level to illustrate the distribution frequency of fluorescence intensity across the entire yeast population. This methodology enables the observation and subsequent discussion of the heterogeneity in EGFP expression.

New comment: not done.

5) Western Blotting: Please explain why SDS-PAGE was selected over native electrophoresis for detecting EGFP, given that it is a fluorescent protein. In light of this approach, the inclusion of an additional 6xHis-tag is unwarranted.

New comment: not done.

New remark: lines 181-197, two new sub-sections were added at the end of the Materials and Methods section. The date on which the site was visited must be specified.

Results section

6) Figure 2: Please indicate the molecular weight of each lane from the DNA ladder.

New comment:  molecular weight are given in B and C, but no information was given in A and D.

7) The data obtained with the empty plasmid are missing from Figures 3, 4, 5, and 6. This control procedure is essential for evaluating the impact of plasmid presence on yeast behavior.

New comment: not done.

8) Figure 4:

Title: Saccharomyces cerevisiae must be written in italics

New comment: line 258 not done.

It should be easier for the reader to specify the lag time, the growth rate and the OD 600 max for each growth curve. It is possible to employ an online software such as Sym’Previus (https://symprevius.eu/en/), to calculate these parameters for each replicate and subsequently perform a statistical analysis.

New comment: not done.

Line 267: please correct the word “fluresens”

9) Figure 5: It is necessary to compare the fluorescence intensity displayed by the mutants of interest with the fluorescence intensity displayed by a reference mutant expressing EGFP to more accurately assess the relevance of the fluorescence intensity (displayed by the mutants of interest).

New comment: not done.

10) Bioinformatics analysis: Does the software builds protein structures based on direct energy minimization?

New comment: well done.

If so, were any additional 3D structures proposed that differ from those shown in the paper?

New comment: not done.

11) Figure 7: The proposed format for the legend is not conducive to rapid identification of the various regions within each sticker. It would be beneficial to include a title for each line or column, such as "EGFP" and "PolyR EGFP," and consider utilizing a single color for a single region, thereby facilitating direct identification of the regions on each model. For example, it is not readily apparent which regions of the models are associated with the 6xHis-tag of the EGFP-PolyR protein. To facilitate comprehension, it would be beneficial to consider analogous color schemes between Figures 1 and 7.

It would be beneficial to include additional information in the figure 7 and 8 legends to assist the reader in comprehending the various figures. Furthermore, the EGFP structure should be annotated as N-terminal and C-terminal. Figure 8: where is the yellow region?

Discussion section

New comment, line 478: Title of the first new sub-section: Saccharomyces cerevisiae must be written in italics.

12) Section entitled “Modified protein plasmid insertion causes a decrease in the growth curve of S. cerevisiae”:

Please verify the following sentence: "Adding raffinose to the growth medium of S. cerevisiae can activate the programmed cell death formation of acetic acid." Indeed, Guaragnella et al. (2013) demonstrated that yeast cells grown on raffinose exhibited enhanced resistance to acetic acid-induced programmed cell death (PCD). However, this observation does not necessarily imply that PCD was actively triggered in the presence of raffinose. The discussion should be more measured, given that the concentration of raffinose used in the Guaragnella et al. (2013) study was two times higher (2%) than that used in the present study (1%). It is therefore pertinent to inquire whether the activation of the retrograde pathway occurred at a lower concentration of raffinose. To reinforce this point, it would be beneficial to cite relevant papers in the field.

New comment: not done.

13) Table 1:

It should be noted that the parameters presented in this table are not discussed in the paper. One might inquire, for instance, how the charges of EGFP and EGFP-PolyR are affected by the intracellular pH in yeast in relation to their pI. It would be beneficial to ascertain whether there are any consequences on EGFP aggregation and fluorescence, for example.  For further insight, please consult the following paper, which will enhance the quality of the discussion:

Nathalia Vieira dos Santos, Carolina Falaschi Saponi, Timothy M. Ryan, Fernando L. Primo, Tamar L. Greaves, Jorge F.B. Pereira. Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors. International Journal of Biological Macromolecules, Volume 164, 2020, Pages 3474-3484. Complementary papers about intracellular pH in yeast could be found to reinforce the discussion.

New comment: not done.

It would be beneficial for the reader to highlight the different regions of the protein sequences with varying colors (see comment 9). Furthermore, providing additional information about the sequence situated between the EGFP and 6xHis-tag in the EGFP sequence (EFCRYPAQWRPLESRGPFEGKPIPNPLLGLDSTRT) and the sequence between the EGFP and PolyR-6xHis-tag sequences in the EGFP-PolyR sequence (EFESGGGGSPG) would be beneficial. Why did the authors consider these two different “linkers”?

New comment: not done.

14) The discussion about the effect of the presence of the EGFP on growth could be enhanced by considering the implementation of supplementary experiments with the objective of elucidating its impact on specific physiological characteristics, such as mitochondrial and metabolic activities. The utilization of fluorescent microscopy (or flow cytometry) in conjunction with suitable fluorescent probes could be a viable avenue for exploration.

New comment: not done.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Yeast engineering, GFP expression in yeast, fluorescence microscopy, microbial growth, biochemistry, microbial food processes

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.163692.r300366 Reviewer response for version 2 Huang Yide 1 Referee Fujian Normal University, Fuzhou, Fujian, China

Competing interests: No competing interests were disclosed.

 24 7 2024 Copyright: © 2024 Huang Y 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The authors attempted to express EGFP and EGFP-polyR proteins with a 6×His tag in Saccharomyces cerevisiae for subsequent protein transduction studies. They synthesized EGFP and EGFP-polyR sequences, cloned them into the S. cerevisiae expression vector pYES2CT, and introduced the vector into the yeast S. cerevisiae INVSc1 to achieve the expression of EGFP and EGFP-polyR.

However, there are several issues with the manuscript:

1. Figure 1 shows the process of constructing recombinant plasmids, which is part of the methods section, not the results. It is recommended to move it to the methods section under "Plasmid design and construction."

2. The title of the figure legend should provide a summary description of the figure, not a description of the methods.

3. For Figure 4, it is recommended to combine panels A, B, and C into one figure and perform statistical comparisons. This will make it clearer for readers to distinguish the differences. Figure 4D is just another presentation of panels A, B, and C and constitutes redundant data, so it should be removed.

4. In Figure 5, the EGFP intensity observed in panel A at 72 hours is much stronger in the EGFP vector group compared to the EGFP-PolyA vector group. However, the quantitative results in panel B show no difference, indicating that the quantification method used in the manuscript is not accurate. It is suggested to use flow cytometry for detection and quantitative analysis.

5. The authors analyzed the expression of EGFP and EGFP-polyR in the yeast cells using Western blot. However, no distinct bands were observed, and the signal above the expected target band appears diffuse, likely due to non-specific binding of the His-tag antibody. Since EGFP antibodies are more specific than His-tag antibodies, it is recommended to use EGFP antibodies for detection.

6. There are still some mistypes in the manuscript that need to be corrected.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Pichia pastoris, Recombinant expression, Protein engineering, Applied microbiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.163692.r300364 Reviewer response for version 2 Guyot Stéphane 1 Referee https://orcid.org/0000-0002-2316-234X UMR Procédés Alimentaires et Microbiologiques, L'Institut Agro Dijon, Dijon, Bourgogne-Franche-Comté, France

Competing interests: No competing interests were disclosed.

 23 7 2024 Copyright: © 2024 Guyot S 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The main aim of this paper is to propose a method for expressing a EGFP carrying C-terminal PolyR plus 6xHis-tag in the diploid Saccharomyces cerevisiae INVSc1 strain. Such an engineering approach is proposed to study and improve the transduction process that can be used for cell reprogramming to produce pluripotent stem cells. In general, the paper presents the different stages of yeast transformation by insertion of an appropriately modified plasmid, the effect of the presence of EGFP flanked or not by a PolyR sequence on yeast growth and a protein structure prediction of the EGFP tagged or not with PolyR and 6xHis tag. At this stage, there may be room for improvement in the paper. Some suggestions for enhancements are presented below for your consideration.

Major comment

1) My main concern is the functionality of the proteins expressed in Saccharomyces cerevisiae. It is also a question of looking beyond the main objective of this study, which is to optimize the expression of these proteins. Although it would be tedious to carry out an experimental approach involving the cultivation of animal or human cells, I think that in the context of this study it would be interesting to ask whether post-translational modifications made by yeast, which cannot be detected by Western blotting, alter the desired functionality of modified EGFP (PolyR with or without 6xHis-tag) without affecting its fluorescence properties? To validate the present study, it would be advisable to extract the modified EGFP protein, bring it into contact with a human cell, and monitor its internalization (protein transduction) by fluorescence microscopy.

Introduction section

2) From my point of view, the introductory section could be improved by not focusing only on one application based on stem cells, since there are a wide range of applications related to the use of protein transduction in various fields such as therapeutic delivery (drug and gene therapy), molecular biology research, regenerative medicine (stem cell engineering and tissue repair), vaccine development, diagnostics, and neuroscience.

Methods section

3) Centrifugation steps: Please indicate the relative centrifugal force (x g) rather than the revolutions per minute (in rpm) throughout the paper.

4) Fluorescent Microscopy: Please clarify why yeasts were fixed with formaldehyde and not observed under a living state.

Please indicate the version of the ImageJ software and the plugins utilized for the measurement of fluorescence intensity. As the distribution of fluorescence intensity is not uniform throughout the yeast, it is necessary to specify how the analysis was conducted. In particular, it is important to ascertain which pixels were taken into account. Please clarify whether the ratio of fluorescence intensity to yeast was taken into account. Please indicate the number of cells that were counted. The optimal approach is to conduct an analysis at the single-cell level to illustrate the distribution frequency of fluorescence intensity across the entire yeast population. This methodology enables the observation and subsequent discussion of the heterogeneity in EGFP expression.

5) Western Blotting: Please explain why SDS-PAGE was selected over native electrophoresis for detecting EGFP, given that it is a fluorescent protein. In light of this approach, the inclusion of an additional 6xHis-tag is unwarranted.

Results section

6) Figure 2: Please indicate the molecular weight of each lane from the DNA ladder.

7) The data obtained with the empty plasmid are missing from Figures 3, 4, 5, and 6. This control procedure is essential for evaluating the impact of plasmid presence on yeast behavior.

8) Figure 4:

Title: Saccharomyces cerevisiae should be written with in italics

It should be easier for the reader to specify the lag time, the growth rate and the OD 600 max for each growth curve. It is possible to employ an online software such as Sym’Previus (https://symprevius.eu/en/), to calculate these parameters for each replicate and subsequently perform a statistical analysis.

9) Figure 5: It is necessary to compare the fluorescence intensity displayed by the mutants of interest with the fluorescence intensity displayed by a reference mutant expressing EGFP to more accurately assess the relevance of the fluorescence intensity (displayed by the mutants of interest).

10) Bioinformatics analysis: Does the software builds protein structures based on direct energy minimization? If so, were any additional 3D structures proposed that differ from those shown in the paper?

11) Figure 7: The proposed format for the legend is not conducive to rapid identification of the various regions within each sticker. It would be beneficial to include a title for each line or column, such as "EGFP" and "PolyR EGFP," and consider utilizing a single color for a single region, thereby facilitating direct identification of the regions on each model. For example, it is not readily apparent which regions of the models are associated with the 6xHis-tag of the EGFP-PolyR protein. To facilitate comprehension, it would be beneficial to consider analogous color schemes between Figures 1 and 7.

Discussion section

12) Section entitled “Modified protein plasmid insertion causes a decrease in the growth curve of S. cerevisiae”:

Please verify the following sentence: "Adding raffinose to the growth medium of S. cerevisiae can activate the programmed cell death formation of acetic acid." Indeed, Guaragnella et al. (2013) demonstrated that yeast cells grown on raffinose exhibited enhanced resistance to acetic acid-induced programmed cell death (PCD). However, this observation does not necessarily imply that PCD was actively triggered in the presence of raffinose. The discussion should be more measured, given that the concentration of raffinose used in the Guaragnella et al. (2013) study was two times higher (2%) than that used in the present study (1%). It is therefore pertinent to inquire whether the activation of the retrograde pathway occurred at a lower concentration of raffinose. To reinforce this point, it would be beneficial to cite relevant papers in the field.

13) Table 1:

It should be noted that the parameters presented in this table are not discussed in the paper. One might inquire, for instance, how the charges of EGFP and EGFP-PolyR are affected by the intracellular pH in yeast in relation to their pI. It would be beneficial to ascertain whether there are any consequences on EGFP aggregation and fluorescence, for example.  For further insight, please consult the following paper, which will enhance the quality of the discussion:

Nathalia Vieira dos Santos, Carolina Falaschi Saponi, Timothy M. Ryan, Fernando L. Primo, Tamar L. Greaves, Jorge F.B. Pereira. Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors. International Journal of Biological Macromolecules, Volume 164, 2020, Pages 3474-3484. Complementary papers about intracellular pH in yeast could be found to reinforce the discussion.

It would be beneficial for the reader to highlight the different regions of the protein sequences with varying colors (see comment 9). Furthermore, providing additional information about the sequence situated between the EGFP and 6xHis-tag in the EGFP sequence (EFCRYPAQWRPLESRGPFEGKPIPNPLLGLDSTRT) and the sequence between the EGFP and PolyR-6xHis-tag sequences in the EGFP-PolyR sequence (EFESGGGGSPG) would be beneficial. Why did the authors consider these two different “linkers”?

14) The discussion about the effect of the presence of the EGFP on growth could be enhanced by considering the implementation of supplementary experiments with the objective of elucidating its impact on specific physiological characteristics, such as mitochondrial and metabolic activities. The utilization of fluorescent microscopy (or flow cytometry) in conjunction with suitable fluorescent probes could be a viable avenue for exploration.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Yeast engineering, GFP expression in yeast, fluorescence microscopy, microbial growth, biochemistry, microbial food processes

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References : Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors. Int J Biol Macromol .2020;164: 10.1016/j.ijbiomac.2020.08.224 3474-3484 32882278 10.1016/j.ijbiomac.2020.08.224 10.5256/f1000research.135264.r177225 Reviewer response for version 1 Moriya Hisao 1 Referee https://orcid.org/0000-0001-7638-3640 Okayama University, Okayama, Japan

Competing interests: No competing interests were disclosed.

 6 11 2023 Copyright: © 2023 Moriya H 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

In this paper, the authors attempted to express EGFP-polyR in S. cerevisiae. They concluded that EGFP was successfully expressed and that the addition of polyR had no effect on the expression level of EGFP.

However, the expression level of EGFP in S. cerevisiae seems to be much lower than the high expression level we know, and there are many possible improvements. Since many papers have been published on heterologous expression of fluorescent proteins in yeast, the authors should objectively judge how their level of technology compares with them. For example, our gTOW vector system can "express EGFP to the limit," and in that case it can express enough EGFP to be recognized by SDS-PAGE in total protein without Western blotting 1 , 2 .

Codon optimization is also mentioned by the authors. On the other hand, most yeast researchers, including us, use yEGFP3, a codon-optimized EGFP that maximizes expression in yeast 3 , which the authors should try first.

In addition, for plasmid construction, you may want to consider introducing a strong homologous recombination cloning method, which is available in yeast 4 . Cell disruption by glass beads is also easier and less expensive than spheroplasting in yeast. In our experience, EGFP-his6 can be purified in large quantities with virtually no degradation. In fact, we have been using the above plasmid to express and purify yEGFP-his6 by glass bead disruption as a practical training for students. If necessary, the plasmid can be distributed by us.

Finally, polyR is known to stall translation 5 , a finding that may be helpful when trying to express EGFP-polyR after successful mass expression of EGFP.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Yeast, overexpression, plasmid construction, systems biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References : Cellular growth defects triggered by an overload of protein localization processes. Sci Rep .2016;6: 10.1038/srep31774 31774 27538565 10.1038/srep31774 : Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome. G3 (Bethesda) .2022;12(6) : 10.1093/g3journal/jkac106 35485947 10.1093/g3journal/jkac106 : Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology (Reading) .1997;143 ( Pt 2): 10.1099/00221287-143-2-303 303-311 9043107 10.1099/00221287-143-2-303 : Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res .1997;25(2) : 10.1093/nar/25.2.451 451-2 9016579 10.1093/nar/25.2.451 : Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate. J Biol Chem .2023;299(1) : 10.1016/j.jbc.2022.102774 102774 36481270 10.1016/j.jbc.2022.102774 10.5256/f1000research.135264.r159002 Reviewer response for version 1 Hasan Khomaini 1 Referee https://orcid.org/0000-0002-7395-4206 Faculty of Medicine, Universitas Jenderal Achmad Yani, Cimahi, Indonesia

Competing interests: No competing interests were disclosed.

 3 11 2023 Copyright: © 2023 Hasan K 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

Pramono and Co-workers study on expression of recombinant Enhanced Green Fluorescent Protein (EGFP) with some modification with the addition of poly-R in order to help protein penetration within cells. In general, the work can be significantly improved, especially in terms of quality of the data due to lack of evidence that are correlated with the conclusions. 

First, as we read in the article, Pramono and co-workers were struggling on how to express and obtained the recombinant protein in sufficient amount. Even though, the Western Blot microscopic data which were presented in the manuscript confirms the success of protein expression, but the quality of SDS PAGE gels did not fully support their conclusion. The gel itself lacks some data and low quality, for instance, no clear molecular weight marker and the bands, which were considered as expressed proteins, were almost invisible. 

Second, the quantitative methodology, which were conducted and presented here, is mostly not effective to be done, because most of them show unexpected results. For instance, when they claimed that the expressed protein was low, they did ultrafiltration with 5 kDa cutoff and resulted no significant improvement. They also failed to prove that additional 6-his-tag in the recombinant protein is useful for their experiment as well. In order to answer our curiosity on the 6-histag, I would suggest to do a protein purification, so the claim of structure model that were presented in the manuscript can be proven and relevant.

Third, the claim on the effect of plasmid toward growth of the yeast is weak due to lack of repetition to the experiment. If the experiment is conducted triplicates, the potential bias can be reduced and the issue of reproducibility may not occur. Additionally, the genetic optimization which was conducted computationally are useless due to having no experimental data that support the claim. 

Fourth, the quality of the figures is very poor in quality. The authors can improve by using a camera or similar equipment that meets the requirement of the quality.

Lastly, some mistypes can be prevented.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Protein chemistry, protein engineering, enzymology, protein therapeutics, biocatalysis and biotransformation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.