Fusion with heat-resistant obscure (Hero) proteins have the potential to improve the molecular property of recombinant proteins

  1. Eri Morimoto
  2. Kotaro Tsuboyama
  3. Yukihide Tomari

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Abstract

Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other “client” proteins in trans from various stresses in vitro and in vivo . Here, we show that fusion of Hero proteins in cis can enhance the molecular property of recombinant proteins. Fusion with Hero11 improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli . Moreover, fusing with Hero9 strongly protected the activity of firefly luciferase bearing destabilizing mutations against heat and other stress conditions. These data suggest that Hero proteins have the potential to be used as versatile stabilization tags for recombinant protein production.

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  1. ASAPbio crowd review

    Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Oana Nicoleta Antonescu, Ruchika Bajaj, Sree Rama Chaitanya and Akihito Inoue. Review synthesized by Ruchika Bajaj.

    This study has characterized the function of Hero proteins in improving the recombinant expression of TAR DNA-binding protein in E. coli and restoration of enzymatic activity of firefly luciferase during heat and stress conditions. This study may be useful for future applications of Hero proteins in life sciences research. Please see below a few points offered as suggestions to help improve the study.

    • In introduction, 3rd paragraph, in context with “amino acid composition and length of Hero proteins”, please elaborate on the effect of these two factors on the function and stability of hero proteins.
    • The manuscript refers to “cis and trans” terms on several occasions. Please explain these terms in context with the association of Hero protein with the target proteins.
    • Introduction - A paragraph describing the origin of Hero proteins and the differences between the types of Hero proteins in the introduction section would be helpful for readers to understand the background on these proteins. For example, please explain the background on naming these proteins as Hero 7, 9, 11 etc. The genes SERF2, C9orf16, C19orf53, etc are mentioned in the plasmid construction section in the Material and methods. Please provide a brief explanation for the relationship between these genes and Hero proteins.
    • Please add more details in the Material and methods section, especifically in western blotting and the luciferase assay, to support the reproducibility of these experiments.
    • Figure 1A. Please explain the role of each component (for example factorXa) either in the text or the legend.
    • Figure 1B: Please add clarification regarding the normalization of lanes by total protein concentration.
    • Fig 1C. Please provide an explanation for the higher order bands in the western blot. The western blot using anti-FLAG antibodies shows non-specific bands. Alternative tags or antibodies or detection methods may be used, for example, GFP tag and in-gel fluorescence can be used to check the expression.
    • Figure 1D and 1E, the error bars are high. Suggest checking the data and providing the mathematical expressions used to calculate relative yields.
    • Figure 2D and E, the error bars are high, access to the raw data behind the graphs may aid interpretation. An explanation for the choice of temperatures 33 C and 37 C would be helpful. Is there any relation between the choice of temperature and the Tm of the protein? The protein is directly being treated at high temperature, similar experiments with cell-based assays would be helpful to understand the effect of the Hero proteins on the stability of Fluc. Would it be possible to report the mathematical expressions used to calculate “Remaining Fluc activity”. Recommend indicating n if these activities are calculated per mg of the protein. Please explain if the reduction in activity is due to loss of protein or loss of luminescence activity from each molecule of the protein.
    • Figure S1, access to the raw data would be helpful to understand the signal to noise ratio for activity.
    • Figure 2 and 3 show similar experiments with wild type and mutants, it may be possible to combine the figures (for example, to avoid the redundancy in Figure 2C and 3A).
    • Figure 3D and G, access to the raw data would be helpful to interpret the signal and noise ratio especially given the low values.
    • Figure 4, Can some further discussion be provided for the reason for higher residual activity for SM and DM than wild type? Tm experiments during stress conditions (heat shock and freeze thaw cycles) may be helpful to define the stability of Fluc and Fluc mutants.
    • Figure 5: Suggest including an explanation for choosing Proteinase K -among other proteases- for these experiments.
    • The residual activity is different in Figure 4 and 5, which could be due to different stress conditions. Please include some discussion about possible explanations.
    • In section “Hero proteins protect Fluc activity better in cis than in trans”, ‘When the molarity of recombinant GST, Hero9, and Hero11 proteins was increased by 10-fold...’ does molarity refer to the concentration of protein ?
    • In the first paragraph of the discussion, “physical shield that prevents collisions of molecules leading to denaturation” and “maintaining the proper folding” is mentioned. Is it the hypothesis for the mechanism behind the stability provided by Hero proteins? Can further discussion on this be provided, along with a relevant reference.
    • In the discussion section, it is mentioned that “Hero may be reminiscent of polyethylene glycol (PEG)”. Please provide further explanation for why hero proteins are correlated with PEG in this fragment.
    • A discussion on why specific Hero proteins may be better for specific target proteins may be helpful.
    • In the second paragraph, of the Discussion “Hero protein can behave differently depending on the client protein and condition” and “important to test multiple Hero proteins to identify one that best protects the protein of interest” are mentioned. Suggest adding further discussion of these points, for example around any alternatives or computational predictions or simulations to test individual Hero proteins for specific client proteins.
  2. Version published to 10.1371/journal.pone.0270097
  3. Version published to 10.1101/2022.03.03.482928v2 on bioRxiv
  4. Version published to 10.1101/2022.03.03.482928v1 on bioRxiv