In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system.
Synthetic DNA libraries encoding human antibody V(L) and V(H) fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >10(10). To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established.
Shibui
Tatsuro
T
MOLECUENCE Corporation, Mitsubishi, Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, 227-8502, Japan. 1807766@cc.m-kagaku.co.jp
Kobayashi
Teruaki
T
Kanatani
Keiichiro
K
Koga
Hirohisa
H
Misawa
Satoru
S
Isomura
Tetsu
T
Sasaki
Tooru
T
D000595:Amino Acid Sequence; D002457:Cell Extracts; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014908:Triticum
D002457:Cell Extracts; D007135:Immunoglobulin Variable Region; D009841:Oligonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins