Review Reports - Plant-Derived Hydrolysates Are a Suitable Replacement for Tryptone N1 in Recombinant Protein Expression Using Human Embryonic Kidney (HEK293-6E) Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an important practical question in recombinant protein production: whether plant-derived hydrolysates can substitute for the commonly used animal-derived Tryptone N1 (TN1) in HEK293 expression systems. The study is clearly motivated, given regulatory constraints on animal by-product transport and the growing interest in sustainable bioprocessing. The experimental approach—comparing pea- and broad-bean–derived hydrolysates with TN1 and a no-hydrolysate control in a PEI-mediated transient transfection workflow—is appropriate and generally well executed.

Although the manuscript is well-written, there are some minor comments that need to be corrected.

The supplementary figures need to be labelled properly and mentioned in the figure legend.

Author Response

Reviewer #1

The supplementary figures need to be labelled properly and mentioned in the figure legend.

Thank you for pointing this out. We have now checked and ensured that all supplementary Figures are mentioned in the Text, including a new Supplementary Figure S1 showing an example of the GFP transfection requested by the other two reviewers.

Reviewer 2 Report

Comments and Suggestions for Authors

This study aims to identify an alternative to TrypTone N1 for recombinant protein expression. The following changes can be incorporated:

1) Which gene or protein is targeted during transfection?

2) Consider adding microscopic images of cells with GFP Signal

3) Was a whole cell extract, nuclear, or cytosolic content used for Western Blot? How much concentration of cell extract was loaded for each well? The internal control is needed to compare the Western blot bands .

4)Why is Tryptone N1 the most commonly used hydrolysate in recombinant protein expression? Mention its advantages

Author Response

Reviewer #2

Comments and Suggestions for Authors

This study aims to identify an alternative to Tryptone N1 for recombinant protein expression. The following changes can be incorporated:

Which gene or protein is targeted during transfection?

The plasmid contains the cDNA sequence encoding an Echinococcus granulosus tegumental antigen (UniProt ID: U6JFD4) fused with a C-terminal Myc and His-8 tag encoded in pTT28. This is mentioned in Lines 91-92 in the formatted manuscript. Interested readers can look up the UniProt ID.

Consider adding microscopic images of cells with GFP Signal

This was also requested by Reviewer #3 (see comments to remark Nr. 2). A new Figure S1 has been added to the supplementary data.

Was a whole cell extract, nuclear, or cytosolic content used for Western Blot? How much concentration of cell extract was loaded for each well? The internal control is needed to compare the Western blot bands.

No extracts were used for Western Blotting. Recombinant expression was using a serum-free medium and the recombinant protein harvested from the supernatant We have added a line to clarify that the plasmid adds a secretors signal sequence (Lines 93-94). As described in Lines 120-125, the recombinant protein was concentrated using TCA precipitation. We used total lane quantification which has been developed as superior alternative to the is more error-prone use of internal controls. In fact, in our approach it would not be possible to use any internal control, as the main protein contained in the supernatant from serum-free medium is the expressed protein, with some minor component resulting from proteins released by the cells during growth or after cell death.

4)Why is Tryptone N1 the most commonly used hydrolysate in recombinant protein expression? Mention its advantages

We have added a whole new section to the discussion (lines 256-262) explaining the advantages which have led to TN1 becoming the standard hydrolysate used for transient recombinant expression in HEK293 cells.

Reviewer 3 Report

Comments and Suggestions for Authors

The work of Shabir et al., investigates the possibility to use Plant-Derived Hydrolysates in order to replace the animal-derived Tryptone N1 (TN1) hydrolysate that is used to improve the expression levels of proteins in HEK293-6E Cells.

The manuscript is descriptive, well written and easy to follow. I just have a few comments that could improve the manuscript:

In all the figures including protein samples (i.e. Fig 2, Fig S2, Fig S3, line 348, Figure S5) the authors should include information about the amount of protein used and not refer to dilution factors and μL loaded. Please use the amount loaded in each lane in μg (or mg) so that anyone can repeat the experiments. As presented, it is not helpful at all for the readers of this study to understand how much protein you used in each case. This information should be included also in the Materials and Methods Section. For example, "300 μL of TCA was mixed with 1200 μL of protein sample" (line 118) how many mg of protein is this?

Line 160: "The transfection efficiency was monitored by transfection of a GFP-encoding plasmid in parallel". It would be useful for the readers of this study to include also some representative pictures obtained from the fluorescent microscope mentioned (line 98). Also include the necessary information in the Methods Section (i.e. microscope model, filters used, camera used for capturing the images).

Figure 1: In the biological replicates A and C, the cell density in the case of negative control (NC) is more than four times less compared to the other three treatments. In addition, the Stain-free blot images in Figure S5, shows that in the case of NC the total protein loaded is also much lower compared to the other treatments. The authors should comment on this within the manuscript and discuss that possibly the lower amount of signal detected in the corresponding Western blots developed with an Anti-his-tag may be due to less amount of protein loaded in the wells and not due to lower transfection efficiency.

Figure 2: "A" & "B" should be aligned properly in the inset of the two panels.

line 254: "less likely to affected". Please rephrase

Author Response

Reviewer #3

The manuscript is descriptive, well written and easy to follow. I just have a few comments that could improve the manuscript:

We thank this reviewer for the positive comments and have addressed the comments as below. We have numbered the suggestions for convenience.

In all the figures including protein samples (i.e. Fig 2, Fig S2, Fig S3, line 348, Figure S5) the authors should include information about the amount of protein used and not refer to dilution factors and μL loaded. Please use the amount loaded in each lane in μg (or mg) so that anyone can repeat the experiments. As presented, it is not helpful at all for the readers of this study to understand how much protein you used in each case. This information should be included also in the Materials and Methods Section. For example, "300 μL of TCA was mixed with 1200 μL of protein sample" (line 118) how many mg of protein is this?

The chosen approach of total lane quantification consists in normalizing the data based on the total protein content in each lane. Protein yields, and therefore the amounts of protein loaded, will depend on the identity of the protein being expressed. In our case, we chose Echinococcus granulosus (UniProt ID: U6JFD4) protein that is not easy to express in large amounts. The described approach based on serial dilutions was also employed by Taylor and colleagues, who established the method published in Mol Biotechnol (2013) 55:217–226 (see Figures 2 and 3). However, Taylor et al. also give loaded protein amounts as they used HeLa cell lysates (in fact, the authors used the much easier approach of spiking a HeLa cell lysate with a purified protein). In contrast, we retrieved our recombinant protein from the serum-free supernatant of the cell cultures. Due to the choice of a difficult to express protein, the protein concentrations in the supernatant are very low, in most cases below the detection limit of protein concentration tests such as BCA with a typical detection limit of 20 µg/mL. Sensitivity of course is not a problem when using chemiluminescent detection, but also there the use of twofold serial dilutions poses no problem.

We actually believe that the method as it is described here, using serial dilutions of the pooled precipitated samples (lines 129-130) for determination of linear range of the antibodies is highly applicable to any protein expressed, independently of the concentrations used. In other words, our method makes the described technique independent of the actual starting protein concentration and is therefore applicable to most settings – i.e. it enhances rather than diminishes reproducibility. Finally, in our study, we only compared relative changes in expression of recombinant protein expression, to demonstrate that using plant-derived peptones does not affect expression negatively - we were not interested in absolute yields.

Line 160: "The transfection efficiency was monitored by transfection of a GFP-encoding plasmid in parallel". It would be useful for the readers of this study to include also some representative pictures obtained from the fluorescent microscope mentioned (line 98). Also include the necessary information in the Methods Section (i.e. microscope model, filters used, camera used for capturing the images).

We have added a Figure (Fig S2) to the supplementary data showing fluorescence obtained 24 hour after transfection and description of the equipment used in the Materials and methods section (Lines 98-99). However, as discussed in the text (lines 237-241), the actual transfection efficiency is not relevant to the results obtained as all cells within one experiment were obtained from a single large scale transfection, equally split into 12 flasks. Furthermore, strictly speaking, the GFP transfection only informs about the transfection of the cells which were transfected with the GFP-encoding plasmid, not the cells transfected with the plasmid encoding our gene of interest, which was a separate transfection. To obtain an accurate percentage of transfection, it would have been necessary to e.g. covalently label the plasmid fluorescently and assess the percentage of fluorescent cells and the mean fluorescence intensity after transfection using flow cytometry. Such techniques exist but were not deemed necessary as we used the same transfected cell population for each experiment (lines 101-104, 236-41). The main reason why we included GFP transfection was as quality check for the integrity of the transfection reagent, as GFP results can be easily seen already after 24 hours, instead of 5 days later when checking for recombinant expression.

Figure 1: In the biological replicates A and C, the cell density in the case of negative control (NC) is more than four times less compared to the other three treatments. In addition, the Stain-free blot images in Figure S5, shows that in the case of NC the total protein loaded is also much lower compared to the other treatments. The authors should comment on this within the manuscript and discuss that possibly the lower amount of signal detected in the corresponding Western blots developed with an Anti-his-tag may be due to less amount of protein loaded in the wells and not due to lower transfection efficiency.

While there are no measurable differences in viable cell counts after 24 hours, the absence of supplementation (NC) results in partially compromised cell viability after 96 hours, and high cell death after 120 hours. However, in order to be able to draw more firm conclusions about the relationship between cell viability and protein yields, we would have needed a different experimental approach. Based on our data, it is reasonable to assume that the different treatments do not affect the cell viability in the first 72 hours, after which the discrepancy between the supplemented groups and the non-supplemented starts becoming visible. However, recombinant protein expression was assessed only once after 120 hours, representing the sum of all protein expressed at any time during the five days of culture. Although cell viability and recombinant protein expression are tightly coupled, their relationship is non-linear and dependent on many factors. We did not determine the specific productivity, defined as recombinant protein produced per viable cell per time. The total yield will ultimately depend on the viable cell density, the specific productivity and the culture time. However, we are very confident that none of the differences are a consequence of transfection efficiencies, as all cells within one experiment were derived from a single large batch of transfected cells (see lines 235-241). Also, transfection efficiencies were around 30% between experimental repeats A, B and C, ruling out this parameter as a source of difference.

Figure 2: "A" & "B" should be aligned properly in the inset of the two panels.

We trust that issues of alignment will be fixed by the production team of the Journal and will check that this is the case in the galley proofs.

line 254: "less likely to affected". Please rephrase

We have added the missing verb, the sentence has been rephrased to “less likely to be affected”.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for the responses. The microscopic image of tranfected HEK293-6E cells with pTTo-GFPq seems inappropriate to me.

Author Response

Thanks for the responses. The microscopic image of transfected HEK293-6E cells with pTTo-GFPq seems inappropriate to me.

Answer - we agree. Images were taken solely for the purpose of confirming that the transfection reagents are still working (quality control), as explained in the text. All treatments were from the same transfected batch of cells, ruling out transfection efficiency as a variable. GFP-transfected cells show a very wide range of fluorescence intensities, ranging from very low (but still visible) fluorescence to what we like to call 'supernova' fluorescence intensity. This huge dynamic range is difficult to capture with our fluorescent microscope. We have therefore removed the Figure which had been requested by one of the reviewers.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The paper can be accepted in the current form.