An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.
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.
Tabuchi
Ichiro
I
Department of Functional Materials Science, Saitama University. 255 Shimo-okubo, Saitama 338-857. Japan.; GenCom Co. 11 Minami-Oya, Machida 194-8511. Japan.Present address: Tokyo Evolution Research Center. 1-1-45-504, Okubo, Shinjuku-ku, Tokyo, 169-0072. Japan. husimi@fms.saitama-u.ac.jp
Soramoto
Sayaka
S
Suzuki
Miho
M
Nishigaki
Koichi
K
Nemoto
Naoto
N
Husimi
Yuzuru
Y