Enzymatic protein fusions with 100% product yield

Reviewer #1 (Public review):

Fuchs describes a novel method of enzymatic protein-protein conjugation using the enzyme Connectase. The author is able to make this process irreversible by screening different Connectase recognition sites to find an alternative sequence that is also accepted by the enzyme. They are then able to selectively render the byproduct of the reaction inactive, preventing the reverse reaction, and add the desired conjugate with the alternative recognition sequence to achieve near-complete conversion. I agree with the authors that this novel enzymatic protein fusion method has several applications in the field of bioconjugation, ranging from biophysical assay conduction to therapeutic development. Previously the author has published on the discovery of the Connectase enzymes and has shown its utility in tagging proteins and detecting them by in-gel fluorescence. They now extend their work to include the application of Connectase in creating protein-protein fusions, antibody-protein conjugates, and cyclic/polymerized proteins. As mentioned by the author, enzymatic protein conjugation methods can provide several benefits over other non-specific and click chemistry labeling methods. Connectase specifically can provide some benefits over the more widely used Sortase, depending on the nature of the species that is desired to be conjugated. However, due to a similar lengthy sequence between conjugation partners, the method described in this paper does not provide clear benefits over the existing SpyTag-SpyCatcher conjugation system. Additionally, specific disadvantages of the method described are not thoroughly investigated, such as difficulty in purifying and separating the desired product from the multiple proteins used. Overall, this method provides a novel, reproducible way to enzymatically create protein-protein conjugates.

The manuscript is well-written and will be of interest to those who are specifically working on chemical protein modifications and bioconjugation.

Reviewer #2 (Public review):

Summary:

Unlike previous traditional protein fusion protocols, the author claims their proposed new method is fast, simple, specific, reversible, and results in a complete 1:1 fusion. A multi-disciplinary approach from cloning and purification, biochemical analyses, and proteomic mass spec confirmation revealed fusion products were achieved.

Strengths:

The author provides convincing evidence that an alternative to traditional protein fusion synthesis is more efficient with 100% yields using connectase. The author optimized the protocol's efficiency with assays replacing a single amino acid and identification of a proline aminopeptidase, Bacilius coagulans (BcPAP), as a usable enzyme to use in the fusion reaction. Multiple examples including Ubiquitin, GST, and antibody fusion/conjugations reveal how this method can be applied to a diverse range of biological processes.

Weaknesses:

Though the ~100% ligation efficiency is an advancement, the long recognition linker may be the biggest drawback. For large native proteins that are challenging/cannot be synthesized and require multiple connectase ligation reactions to yield a complete continuous product, the multiple interruptions with long linkers will likely interfere with protein folding, resulting in non-native protein structures. This method will be a good alternative to traditional approaches as the author mentioned but limited to generating epitope/peptide/protein tagged proteins, and not for synthetic protein biology aimed at examining native/endogenous protein function in vitro.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Fuchs describes a novel method of enzymatic protein-protein conjugation using the enzyme Connectase. The author is able to make this process irreversible by screening different Connectase recognition sites to find an alternative sequence that is also accepted by the enzyme. They are then able to selectively render the byproduct of the reaction inactive, preventing the reverse reaction, and add the desired conjugate with the alternative recognition sequence to achieve near-complete conversion. I agree with the authors that this novel enzymatic protein fusion method has several applications in the field of bioconjugation, ranging from biophysical assay conduction to therapeutic development. Previously the author has published on the discovery of the Connectase enzymes and has shown its utility in tagging proteins and detecting them by in-gel fluorescence. They now extend their work to include the application of Connectase in creating protein-protein fusions, antibody-protein conjugates, and cyclic/polymerized proteins. As mentioned by the author, enzymatic protein conjugation methods can provide several benefits over other non-specific and click chemistry labeling methods. Connectase specifically can provide some benefits over the more widely used Sortase, depending on the nature of the species that is desired to be conjugated. However, due to a similar lengthy sequence between conjugation partners, the method described in this paper does not provide clear benefits over the existing SpyTag-SpyCatcher conjugation system. Additionally, specific disadvantages of the method described are not thoroughly investigated, such as difficulty in purifying and separating the desired product from the multiple proteins used. Overall, this method provides a novel, reproducible way to enzymatically create protein-protein conjugates.

The manuscript is well-written and will be of interest to those who are specifically working on chemical protein modifications and bioconjugation.

Reviewer #2 (Public review):

Summary:

Unlike previous traditional protein fusion protocols, the author claims their proposed new method is fast, simple, specific, reversible, and results in a complete 1:1 fusion. A multi-disciplinary approach from cloning and purification, biochemical analyses, and proteomic mass spec confirmation revealed fusion products were achieved.

Strengths:

The author provides convincing evidence that an alternative to traditional protein fusion synthesis is more efficient with 100% yields using connectase. The author optimized the protocol's efficiency with assays replacing a single amino acid and identification of a proline aminopeptidase, Bacilius coagulans (BcPAP), as a usable enzyme to use in the fusion reaction. Multiple examples including Ubiquitin, GST, and antibody fusion/conjugations reveal how this method can be applied to a diverse range of biological processes.

Weaknesses:

Though the ~100% ligation efficiency is an advancement, the long recognition linker may be the biggest drawback. For large native proteins that are challenging/cannot be synthesized and require multiple connectase ligation reactions to yield a complete continuous product, the multiple interruptions with long linkers will likely interfere with protein folding, resulting in non-native protein structures. This method will be a good alternative to traditional approaches as the author mentioned but limited to generating epitope/peptide/protein tagged proteins, and not for synthetic protein biology aimed at examining native/endogenous protein function in vitro.

I would like to sincerely thank both reviewers for their insightful and constructive feedback on the manuscript. I have addressed reviewer #1’s comments below:

(1) The benefits over the SpyTag-SpyCatcher system. Here, the conjugation partners are fused via the 12.3 kDa SpyCatcher protein, which is considerably larger than the Connectase fusion sequence (20 aa). This is briefly mentioned in the introduction (p. 1 ln 24-25). In a related technology, the SpyTag-SpyCatcher system was split into three components, SpyLigase, SpyTag and KTag (Fierer et al., PNAS 2014). The resulting method introduces a sequence between the fusion partners (SpyTag (13aa) + KTag (10aa)), which is similar in length to the Connectase fusion sequence. I mention this method in the discussion (p. 8, ln 296 - 297), but preferred not to comment on its efficiency. It appears to require more enzyme and longer incubation times, while yielding less fusion product (Fierer et al., Figure 2).

(2) Purification of the fusion product. The method is actually advantageous in this respect, as described in the discussion (p. 8, ln 257-263). I plan to add a figure showing an example in the revised article.