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J. Biol. Chem., Vol. 280, Issue 26, 24752-24758, July 1, 2005

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Cellular Quality Control Screening to Identify Amino Acid Pairs for Substituting the Disulfide Bonds in Immunoglobulin Fold Domains^*

Yoshihisa Hagihara, Tomoki Matsuda¶, and Noboru Yumoto||

From the Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan and ^¶Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

We are interested in determining which amino acid pairs can be substituted for the disulfide (S–S) bonds in proteins without disrupting their native structures under physiological conditions. In this study, we focused on the intradomain S–S bonds in Ig fold domains and aimed to determine a simple rule for replacement of their S–S bonds. The cysteines of four different Ig fold domains were mutated randomly, and the amino acid pairs substituted for the S–S bonds were screened by the method utilizing a cellular quality control system. Among the 36 selected mutants, 31 were natively folded without S–S bonds, as judged from the cooperativity of thermal unfolding. In addition, the selected mutant llama heavy chain antibodies retained antigen-binding affinity. At least two of the pairs Ala:Ala, Ala:Val, Val: Ala, and Val:Val were found in the selected mutants for all four different Ig fold domains, and they were stably folded at 30 °C. This suggests that examination of these four pairs could be enough to obtain natively folded Ig fold domains without S–S bonds.

Received for publication, April 12, 2005 , and in revised form, May 2, 2005.

^* This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Support for Young Researchers with a Term). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed. Tel.: 81-72-751-9274; Fax: 81-72-751-9628; E-mail: hagihara-kappael{at}aist.go.jp. || To whom correspondence may be addressed. Tel.: 81-72-751-9521; Fax: 81-72-751-9628; E-mail: n-yumoto{at}aist.go.jp.

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				Y. Hagihara, S. Mine, and K. Uegaki Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region J. Biol. Chem., December 14, 2007; 282(50): 36489 - 36495. [Abstract] [Full Text] [PDF]

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