Improvement of heavy and light chain assembly by modification of heavy chain constant region 1 (CH1): Application for the construction of an anti-paclitaxel fragment antigen-binding (Fab) antibody

Abstract

Formation of disulfide bonds between heavy and light chains is challenge for production of recombinant immunoglobulin G (IgG) and fragment antigen-binding (Fab). Insufficient formation of the bond influences productive yield and quality of recombinant IgG and Fab. To investigate how amino acid sequences of the first heavy chain constant region (CH1) effect on assembly between heavy chain (VH-CH1 or Fd) and light chain (VL-CL), the CH1 gene sequence of an anti-paclitaxel fragment antigen-binding (anti-PT Fab IgG2a) was modified using the splicing by overlap extension-polymerase chain reaction (SOE-PCR) to be gene sequences encoding amino acid sequence of IgG1, IgG2b, and IgG3 subtypes. These anti-PT Fabs were expressed in Escherichia coli and silkworm larva expression systems. The efficiency of the assembly between VH-CH1 and VL-CL was evaluated. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis, assembly between heavy of IgG1 subtype and light chain was found to be superior over other subtypes using all tested expression systems. Furthermore, reactivity analysis using an enzyme-linked immunosorbent assay (ELISA) revealed that anti-PT Fab IgG1 subtype showed the highest reactivity against target compound paclitaxel. The folding efficiency and reactivity of the anti-PT Fab was influenced by CH1 amino acid sequence, which raises the possibility that this modification can be applied to improve recombinant Fab production.