Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli.
The affinity of natural antibody (Ka = 8 x 10(6) 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 10(7) and 5.2 x 10(7) 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.
Park
S G
SG
Department of Microbiology, College of Medicine, Pusan, 614-735, Korea.
Lee
J S
JS
Je
E Y
EY
Kim
I J
IJ
Chung
J H
JH
Choi
I H
IH
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D001483:Base Sequence; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D017931:DNA Primers; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen