Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli

Sae Gwang Park, Jae Sook Lee, Eun Young Je, Ik Jung Kim, Junho Chung, In Hak Choi

Research output: Contribution to journalArticleResearchpeer-review

18 Citations (Scopus)

Abstract

The affinity of natural antibody (Ka = 8 x 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 x 107 and 5.2 x 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. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)553-557
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume275
Issue number2
DOIs
StatePublished - 28 Aug 2000

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Escherichia coli
Isopropyl Thiogalactoside
Genes
Antibodies
Clone Cells
Inclusion Bodies
Gene encoding
Dialysis
Capsid Proteins
Viruses
Antibody Affinity
Fusion reactions
Point Mutation
Hepatitis B virus
Polyacrylamide Gel Electrophoresis
Proteins

Keywords

  • Affinity maturation
  • Chain shuffling
  • Inclusion body
  • PreS1
  • ScFv

Cite this

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title = "Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli",
abstract = "The affinity of natural antibody (Ka = 8 x 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 x 107 and 5.2 x 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. (C) 2000 Academic Press.",
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Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli. / Park, Sae Gwang; Lee, Jae Sook; Je, Eun Young; Kim, Ik Jung; Chung, Junho; Choi, In Hak.

In: Biochemical and Biophysical Research Communications, Vol. 275, No. 2, 28.08.2000, p. 553-557.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli

AU - Park, Sae Gwang

AU - Lee, Jae Sook

AU - Je, Eun Young

AU - Kim, Ik Jung

AU - Chung, Junho

AU - Choi, In Hak

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SN - 0006-291X

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