An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
AUTOR(ES)
Tabuchi, Ichiro
FONTE
Biological Procedures Online
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=145556Documentos Relacionados
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