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An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
Ichiro Tabuchi1, Sayaka Soramoto, Miho Suzuki
1Department 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
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.