IngentaConnect Affinity Maturation of Natural Antibody Using a Chain Shuffling T...

Authors: Park S-G.¹; Lee J-S.¹; Je E-Y.¹; Kim I-J.²; Chung J-H.³; Choi I-H.¹

Source: Biochemical and Biophysical Research Communications, Volume 275, Number 2, August 2000 , pp. 553-557(5)

Abstract:

The affinity of natural antibody (Ka = 8 × 10⁶ M^-1) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors. Two separate clones, 1C2 and 1E4, showed affinities of 2.3 × 10⁷ and 5.2 × 10⁷ M^-1, which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone. Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG. The expression level of functional rscFv capable of binding to preS1 reached a peak after 6–10 h (1C2) and 8–10 h (1E4) of IPTG induction, and afterwards decreased gradually. In order to achieve the overexpression of rscFv in E. coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector. RscFvs were overexpressed as cytoplasmic inclusion bodies in E. coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system. The purified recombinant fragments were pure when analyzed by SDS–PAGE and had the predicted size of 34 kDa. Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III. Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro. Copyright 2000 Academic Press.

Keywords: affinity maturation; preS1; ScFv; chain shuffling; inclusion body

Language: English

Document Type: Research article

Affiliations: 1: Department of Microbiology, Inje University, Pusan, 614-735, Korea 2: Department of Microbiology, Dongguk University, Kyungju, 780-140, Korea 3: Department of Biochemistry, Seoul National University, Seoul, 110-460, Korea

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Affinity Maturation of Natural Antibody Using a Chain Shuffling Technique and the Expression of Recombinant Antibodies in Escherichia coli