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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Misfolding-assisted Selection of Stable Protein Variants Using Phage Displays

  • Shin, Jong-Shik (Functional Proteomics Center, Korea Institute of Science and Technology) ;
  • Ryu, Seung-Hyun (Functional Proteomics Center, Korea Institute of Science and Technology) ;
  • Lee, Cheol-Ju (Life Sciences Division, Korea Institute of Science and Technology) ;
  • Yu, Myeong-Hee (Functional Proteomics Center, Korea Institute of Science and Technologyersity)
  • Received : 2005.03.04
  • Accepted : 2005.10.11
  • Published : 2006.01.31
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Abstract

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

Keywords

References

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