Misfolding-assisted selection of stable protein variants using phage displays

Jong Shik Shin, Seung Hyun Ryu, Cheolju Lee, Myeong Hee Yu

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-indueed unfolding, that can be used to select protein variants with improved eonformational stability. To test the efficiency of this strategy, wild-type and two stable variants of α1- antitrypsin (α1AT) 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°C). Once the α1AT moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint (Cm) of the α1AT variant, around 20% of the phage retained binding affinity to anti-α1AT 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 α1AT 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.

Original languageEnglish
Pages (from-to)55-60
Number of pages6
JournalJournal of Biochemistry and Molecular Biology
Volume39
Issue number1
Publication statusPublished - 2006 Jan 1

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Bacteriophages
Display devices
Proteins
Bacteriophage M13
Screening
Protein Unfolding
Guanidine
Dialysis
Urea
Milk
Fusion reactions
Genes
Throughput
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Shin, Jong Shik ; Ryu, Seung Hyun ; Lee, Cheolju ; Yu, Myeong Hee. / Misfolding-assisted selection of stable protein variants using phage displays. In: Journal of Biochemistry and Molecular Biology. 2006 ; Vol. 39, No. 1. pp. 55-60.
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Misfolding-assisted selection of stable protein variants using phage displays. / Shin, Jong Shik; Ryu, Seung Hyun; Lee, Cheolju; Yu, Myeong Hee.

In: Journal of Biochemistry and Molecular Biology, Vol. 39, No. 1, 01.01.2006, p. 55-60.

Research output: Contribution to journalArticle

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