Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation.
Single-chain Fv antibody (scFv) having 2 types of polypeptide linkers with or without rare codons, namely scFv (G(4)S)(3)(R) and scFv No.10 (with rare codons) and scFv (G(4)S)(3) and scFv No.10(NR) (without rare codon), were expressed under controllable conditions in batch and fed-batch fermentation, in order to compare volumetric productivity and specific productivity levels of scFvs as a soluble form. In batch fermentation, volumetric productivity levels of scFv (G(4)S)(3)(R) and scFv No.10, namely scFvs having the rare codon linkers were 3-5 times higher than those of scFvs that had linkers without the rare codon. In fed-batch fermentation controlled by an exponential feeding system, the cell concentrations of the transformants increased with similar specific growth rates (0.1 h(-1)), while the specific productivity levels of scFvs with the rare codon linkers were 1.6 times higher than those of scFvs without the rare codon linkers. These results indicate that the presence of several rare codons in the gene of a polypeptide linker increases soluble amount of scFvs. This might be caused by a temporary decrease in translation speed at the position of the polypeptide linker allowing time for the folding of the V(H) domain and avoiding unfavorable interactions between amino acid residues at the unfolded V(H) and V(L) domains. Higher specific productivity levels of both scFv No. 10 and scFv No. 10(NR) than those of scFv (G(4)S)(3)(R) and (G(4)S)(3) might be caused by difference in stability of the polypeptide linkers on the basis of amino acid sequences. Thus, the rare codon linkers tested in this study will be considerably useful for large-scale production of soluble and active scFvs in fed-batch or continuous fermentations, in which high cell activity can be maintained.
Kumada
Yoichi
Y
Department of Chemistry and Materials Technology, Kyoto Institute of Technology, Hashigami-cho, Sakyo-ku, Kyoto 606-8585, Japan. kumada@chem.kit.ac.jp
Sakan
Yoshinobu
Y
Kajihara
Hideyuki
H
Kihara
Mana
M
Kikuchi
Yasufumi
Y
Yamaji
Hideki
H
Seong
Gi Hun
GH
Katoh
Shigeo
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000906:Antibodies / Q000235:genetics; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D007136:Immunoglobulins / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D000596:Amino Acids; D000906:Antibodies; D003062:Codon; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D010455:Peptides; D011994:Recombinant Proteins