Membrane-Based Micro-Volume Dialysis Method for Rapid and High-Throughput Protein Crystallization

Round 1

Reviewer 1 Report

1) Figure 2: please show the difference between Protocol 1A, 1B and 1C (i.e. the sequence of protein/PEG addition).

2) Please provide data for the diffusion of salt, PEG and protein across the membrane, especially for showing the protein does not go through the membrane (the molacular weight cut-off is only a reference)

3) Comment on the osmotic pressure at both sides of the membrane and the resulting flow of water across the membrane.

4) About the statement ".... considerable variability in results obtained with 192 protocol 1B, unlike with protocol 1A which was highly reproducible ... ", please provide statistical analysis about the reproducibility of the results obtained with protocol 1A vs those obtained with protocol 1B.

5) Consider performing the experiment where all the precipitants are on one side of the membrane, while the protein solution is on the other side. Will PEG have problem diffusing through the membrane?

Author Response

Reviewer 1

• Figure 2: please show the difference between Protocol 1A, 1B and 1C (i.e. the sequence of protein/PEG addition).

Response: I thank the reviewer for the suggestion. Figure 2 has been modified as suggested.

• Please provide data for the diffusion of salt, PEG and protein across the membrane, especially for showing the protein does not go through the membrane (the molecular weight cut-off is only a reference)

Response: I thank the reviewer for the suggestion. A discussion on diffusion of salt, PEG and protein has been added in the revised manuscript.

• Comment on the osmotic pressure at both sides of the membrane and the resulting flow of water across the membrane.

Response: I thank the reviewer for the suggestion. A discussion on the osmotic pressure across the membrane and possibility of resultant water flow has been included.

• About the statement ".... considerable variability in results obtained with 192 protocol 1B, unlike with protocol 1A which was highly reproducible ... ", please provide statistical analysis about the reproducibility of the results obtained with protocol 1A vs those obtained with protocol 1B.

Response: I thank the reviewer for the suggestion. The statement ".... considerable variability in results obtained with 192 protocol 1B, unlike with protocol 1A which was highly reproducible ... " has been explained in qualitative terms. Statistical analysis is not possible as there is no quantitative data to analyze.

• Consider performing the experiment where all the precipitants are on one side of the membrane, while the protein solution is on the other side. Will PEG have problem diffusing through the membrane?

Response: I thank the reviewer for the suggestion. However, given that PEG diffuses extremely slowly through the membrane used in this study, the crystallization process would be very slow.

Reviewer 2 Report

While the work is overall well-conducted and insightful, there are a few issues in the manuscript that require clarification and revision to further enhance its quality and impact.

1. As discussed in lines 128-135 and line 176 of the manuscript, the authors should consider including a control experiment that focuses on the crystallization process in the absence of membrane diffusion. Such a control experiment is crucial as it helps ascertain the factors responsible for the observed crystallization process, whether attributable to membrane diffusion, vapor diffusion, or a combination of both.

2. In lines 97-103, the authors present the sequence of protocol 1A in Figure 2. However, the distinction between protocols 1A, 1B, and 1C is not clear. I recommend the authors elaborate and clarify these protocols further for the benefit of the readers.

3. The choice of dialysis membrane and its Molecular Weight Cut-Off (MWCO) should be explained in greater detail. Since the molecular weight of lysozyme is 14.3 kDa, an MWCO of around 7 kDa might be more appropriate. Therefore, it would be helpful if the authors can provide a rationale for their choice of Spectra/Por 4 with an MWCO of 11-14 kDa.

 4. The authors should pay attention to standardizing the format of figures throughout the manuscript. For example, the labels 'A' and 'B' in Figure 6 are positioned in the lower-right corner, but they appear in the upper-left corner in Figure 7. Consistency in the presentation will improve the overall clarity and readability of the manuscript.

Author Response

Reviewer 2

While the work is overall well-conducted and insightful, there are a few issues in the manuscript that require clarification and revision to further enhance its quality and impact.

1. As discussed in lines 128-135 and line 176 of the manuscript, the authors should consider including a control experiment that focuses on the crystallization process in the absence of membrane diffusion. Such a control experiment is crucial as it helps ascertain the factors responsible for the observed crystallization process, whether attributable to membrane diffusion, vapor diffusion, or a combination of both.

Response: I thank the reviewer for the suggestion. When all three components, i.e., the protein and the two crystallizing agents were added on the same side, the crystallization process was not reproducible. Controlled heterogeneity in concentration of the different species is important for crystallization and this is ensured by using the membrane. This has been mentioned in the revised manuscript.

 

2. In lines 97-103, the authors present the sequence of protocol 1A in Figure 2. However, the distinction between protocols 1A, 1B, and 1C is not clear. I recommend the authors elaborate and clarify these protocols further for the benefit of the readers.

Response: I thank the reviewer for the suggestion. A revised Figure 2 showing all three protocols has been included in the revised manuscript.

 

3. The choice of dialysis membrane and its Molecular Weight Cut-Off (MWCO) should be explained in greater detail. Since the molecular weight of lysozyme is 14.3 kDa, an MWCO of around 7 kDa might be more appropriate. Therefore, it would be helpful if the authors can provide a rationale for their choice of Spectra/Por 4 with an MWCO of 11-14 kDa.

Response: I thank the reviewer for the suggestion. The transport of lysozyme through the 11-14 kDa MWCO membrane was negligible. This was assessed using a preliminary experiment. This has been mentioned in the revised manuscript.

 

4. The authors should pay attention to standardizing the format of figures throughout the manuscript. For example, the labels 'A' and 'B' in Figure 6 are positioned in the lower-right corner, but they appear in the upper-left corner in Figure 7. Consistency in the presentation will improve the overall clarity and readability of the manuscript.

Response: I thank the reviewer for the suggestion. However, some of the micrographs have darker and lighter zones. The placement of the labels was influenced by this as putting the labels consistently in one corner would make these difficult to see in some figures.

Reviewer 3 Report

 

The manuscript is a short and succinct study showing dialysis based crystallisation of lysozyme and how the crystallisation process can be influenced by preparing the droplet in such a way as to induce a high degree of concentration heterogeneity in the droplet producing pockets of supersaturation. The manuscript shows that lysozyme crystal growth in be assessed in this set up in the presence of additives. The manuscript is fine in as far as it goes, it represents a simple addition to the crystallisation methodology. However I do feel it is too much to say that this method is suitable for drug development since no drug targets and no drugs are analysed. I strongly suggest the title and scope of the title and introduction are toned down to better reflect the contents of the manuscript.

Author Response

Reviewer 3

The manuscript is a short and succinct study showing dialysis based crystallisation of lysozyme and how the crystallisation process can be influenced by preparing the droplet in such a way as to induce a high degree of concentration heterogeneity in the droplet producing pockets of supersaturation. The manuscript shows that lysozyme crystal growth in be assessed in this set up in the presence of additives. The manuscript is fine in as far as it goes, it represents a simple addition to the crystallisation methodology. However I do feel it is too much to say that this method is suitable for drug development since no drug targets and no drugs are analysed. I strongly suggest the title and scope of the title and introduction are toned down to better reflect the contents of the manuscript.

Response: I thank the reviewer for the suggestion. The scope has been toned down by modifying the title and by mentioning the method could potentially be used for drug screening/development.