Authors
Ichiro Tabuchi, Sayaka Soramoto, Miho Suzuki, Koichi Nishigaki, Naoto Nemoto, Yuzuru Husimi
Summary
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 vitrotranslation. 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 vitrovirus with this “viral genome” was demonstrated.Further details
The protocol was imported from Springer Protocols. To see the entire protocol, click on the source link, or download the pdf here.Advertisement
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