An efficient ligation method in the making of an in vitro virus for in vitro protein evolution |
| |
| Authors: | Ichiro Tabuchi Sayaka Soramoto Miho Suzuki Koichi Nishigaki Naoto Nemoto Yuzuru Husimi |
| |
| Institution: | (1) Department of Functional Materials Science, Saitama University, 255 Shimo-okubo, 338-8570 Saitama, Japan;(2) GenCom Co., 11 Minami-Oya, 194-8511 Machida, Japan;(3) Present address: Tokyo Evolution Research Center, 1-1-45-504, Okubo, Shinjuku-ku, 169-0072 Tokyo, Japan |
| |
| Abstract: | The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion(=viral particle)”(mRNA-peptide fusion),
is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands
ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared
the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification
of the in vitro virus with this “viral genome” was demonstrated.
Published: October 28, 2002 |
| |
| Keywords: | Indexing terms" target="_blank">Indexing terms ligation method evolutionary protein engineering in vitro selection mRNA-protein fusion mRNA display |
| 本文献已被 SpringerLink 等数据库收录！ |
|
