A completely in vitro system for obtaining scFv using mRNA display, PCR, direct sequencing, and wheat embryo cell-free translation.
Using mRNA display followed by in vitro sequencing and translation, a complete in vitro system for obtaining scFv has been developed. An mRNA display library for synthetic scFv was panned against human TNF receptor (TNFR). The nucleotide portion of the enriched molecules was subjected to limiting dilution, and PCR-amplified. Three of the proteins encoded by the amplified fragments were synthesized in a wheat embryo (WE) cell-free system using a batch method. They were shown to bind TNFR by ELISA. One of their sequences was identified in vitro. The identified clone was further synthesized at approx. 0.5 mg/ml reaction mixture in a WE system with dialysis as a totally soluble protein.
Shibui
Tatsuro
T
Molecuence Corp., 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
D000818:Animals; D002457:Cell Extracts / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology; D014908:Triticum / Q000378:metabolism*
D002457:Cell Extracts; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor