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Research Article
Revised

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

[version 2; peer review: 4 not approved]
PUBLISHED 27 Mar 2024
Author details Author details
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This article is included in the Cell & Molecular Biology gateway.

Abstract

Background

The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody.

Methods

The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins.

Results

The addition of a PolyR group to the C-terminal of EGFP carrying C-terminal 6×His-tag showed similar fluorescence expression levels compare to EGFP without addition of PolyR as well. Moreover, yeast with plasmid insertion showed decrease S. cerevisiae growth curve but still preserving the fluorescence EGFP.

Conclusions

The expression of the EGFP modified protein in S. cerevisiae is not affected by the addition of arginine.

Keywords

Saccharomyces cerevisiae INVSc1 protein expression, EGFP protein, protein transductions.

Revised Amendments from Version 1

In this version, There are several changes and additions to the paper :

  • We added one affiliate because the corresponding author has moved to a new agency
  • We changed the abstract in the results, discussion, and conclusion sections. We only reported this study to the fluorescence expression stage because the protein expression results require more substantial evidence. Therefore, there are several parts that we decided to exclude from this paper, namely:
  • The methodology section, Optimization of protein extraction lysis, is omitted. I also added the data with statistical analysis.
  • In the results section, Figure 4 and Figure 5 were revised, and statistical analysis was added to see significant differences between treatments.
  • Then, the results of the optimization of protein extraction with the lysis procedure are also abolished in this section.
  • We decided to remove the codon optimization section in the discussion section because, according to the reviewer, it was irrelevant enough to be included. Then, the discussion section on protein extraction lysis and the use of lyticase is removed to align with this paper's reporting stages. Also, in this discussion section, we explain the advantages and disadvantages of our system.
  • In conclusion, we amended our conclusions accordingly and limited them to reporting protein expression to the fluorescence observation stage so that other parts of the paper could be revised.
  • In the extended data section, We've changed the figure in figshare and included a new link in this paper.
  • In the reference section, we deleted some references and added some new ones according to the revision of this paper.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

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 analyzes and discusses some of the possible improvements of EGFP and EGFP-PolyR protein expression in S. cerevisiae INVSc1.

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 E. coli 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 Saccharomyces cerevisiae 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 Saccharomyces cerevisiae

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, λex 488 nm and λem 507 nm). Fluorescent intensity was measured using ImageJ software, and delta intensity was calculated as follows:

Intensityt=IntensitytIntensity0
where,

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

Intensity0 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.

Bioinformatics analysis of EGFP-polyR

Amino acid composition and physicochemical properties were analyzed using ProtParam,10 and hydrophilic amino acids were analyzed using ProtScale.10 The isoelectric point was measured using the protein Isoelectric Point Calculator.11 The tertiary structure was modeled via i-Tasser12 and AlphaFold,13,14 and the Protean3D was used for viewing to analyze whether the histidine and polyarginine were located in the outer regions.

Results

Construction of Plasmid pYES2CT-EGFP and pYES2CT-EGFP-PolyR and Transformation into Saccharomyces cerevisiae 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-polyR 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.

748a9724-0e96-436a-bef4-31f629da516c_figure1.gif

Figure 1. 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).

748a9724-0e96-436a-bef4-31f629da516c_figure2.gif

Figure 2. pEGFP-Poly R (lane 3) and pYES2CT (lane 9) were cut with restriction enzymes NotI and BamHI, where pEGFP-Poly R produced egfp-polyR 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).

748a9724-0e96-436a-bef4-31f629da516c_figure3.gif

Figure 3. Positive E. coli TOP10 and S. cerevisiae 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 Saccharomyces cerevisiae 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).

748a9724-0e96-436a-bef4-31f629da516c_figure4.gif

Figure 4. Growth curve comparison of Saccharomyces cerevisiae 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).

748a9724-0e96-436a-bef4-31f629da516c_figure5.gif

Figure 5. The fluorescence expressed by S. cerevisiae 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).

748a9724-0e96-436a-bef4-31f629da516c_figure6.gif

Figure 6. Western blotting of protein expressed at 48 hours post-inoculation to induction medium by S. cerevisiae 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 properties of the proteins were analyzed using ProtParam and Isoelectric Point Calculator, and the results are presented in Table 1.

Table 1. Properties of the proteins as analyzed using ProtParam and Isoelectric Point Calculator.

AA: amino acids; pI: Isoelectric point.

PropertiesEGFPEGFP-PolyR
SequenceMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEFCRYPAQWRPLESRGPFEGKPIPNPLLGLDSTRTGHHHHHHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEFESGGGGSPGRRRRRRRRRRRHHHHHH
Number of AA281267
MW31832.9530444.33
pI5.87.4
Extinction coefficients (M-1 cm-1, at 280 nm measured in water)29005
28880 (reduced)
22015
21890 (reduced)
Estimated half-life30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo)
30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo)
Instability index29.11 (stable)52.99 (unstable)
Aliphatic index74.5270.75
Grand average of hydropathicity (GRAVY)-0.572-0.711

Modeling of the proteins was performed using iTasser, and the result is presented in Figure 7. The models show that in both proteins, EGFP and EGFP-PolyR, the 6xHis-tag is located outside the protein nucleus. Structural alignment using Protean3D software predicted that the presence of PolyR indeed affected the conformation of the fused protein, although not within the EGFP domain itself (Figure 7E and F).

748a9724-0e96-436a-bef4-31f629da516c_figure7.gif

Figure 7. The tertiary structure of EGFP (A and B) and EGFP-PolyR (C and D) proteins with 6×His-tag. Alignment results of the proteins tertiary structures (E and F).

Discussion

The addition of PolyR residues did not affect the expression of EGFP protein

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.15 Polyarginine as CPP also plays an essential role in forming hydrogen bonds with cell membrane protein groups.16

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.17 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.18 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).19

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 only with EGFP, 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.

We must take into account, however, that the number of arginine residues in PolyR (11×R), the fluorescent gene, and the host used in our study differs from the aforementioned studies by Reddington18 and Patel.19 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 S. cerevisiae

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.18 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.19,20 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.21 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.

Conclusions

The expression results of protein EGFP and its PolyR fused counterpart, EGFP-PolyR indicated that the predicted conformational change caused by PolyR addition in EGFP-PolyR did not affect the expression levels, since both proteins were expressed at an equal level.

The growth curve of the transformant S. cerevisiae decreased due to the addition of induction mediums, especially raffinose, although it did not affect the amount of expression of the target-modified protein. It can also be caused by the presence of localization to the intracellular compartment, which is expected to cause cellular defects due to excessive localization processes and growth defects in cells. Moreover, PolyR at the C terminal did not cause the EGFP protein translation and fluorescence expression stall, only the growth curve decreased. Our results showed that this plasmid vectors is a promising GFP-PolyR expression. However, further studies still need to be done to show more complete data.

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Tandio Saputro SS, Wahyunita K, Nurhasanah A et al. Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1 [version 2; peer review: 4 not approved]. F1000Research 2024, 12:1 (https://doi.org/10.12688/f1000research.123181.2)
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Reviewer Report 24 Jul 2024
Yide Huang, Fujian Normal University, Fuzhou, Fujian, China 
Not Approved
VIEWS 14
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 ... Continue reading
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Huang Y. Reviewer Report For: Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1 [version 2; peer review: 4 not approved]. F1000Research 2024, 12:1 (https://doi.org/10.5256/f1000research.163692.r300366)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 23 Jul 2024
Stéphane Guyot, UMR Procédés Alimentaires et Microbiologiques, L'Institut Agro Dijon, Dijon, Bourgogne-Franche-Comté, France 
Not Approved
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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 ... Continue reading
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Guyot S. Reviewer Report For: Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1 [version 2; peer review: 4 not approved]. F1000Research 2024, 12:1 (https://doi.org/10.5256/f1000research.163692.r300364)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 06 Nov 2023
Hisao Moriya, Okayama University, Okayama, Japan 
Not Approved
VIEWS 26
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 ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Moriya H. Reviewer Report For: Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1 [version 2; peer review: 4 not approved]. F1000Research 2024, 12:1 (https://doi.org/10.5256/f1000research.135264.r177225)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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52
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Reviewer Report 03 Nov 2023
Khomaini Hasan, Faculty of Medicine, Universitas Jenderal Achmad Yani, Cimahi, Indonesia 
Not Approved
VIEWS 52
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 ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Hasan K. Reviewer Report For: Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1 [version 2; peer review: 4 not approved]. F1000Research 2024, 12:1 (https://doi.org/10.5256/f1000research.135264.r159002)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 03 Jan 2023
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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