Construction of a synthetic messenger RNA encoding a membrane protein.
We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.
D000818:Animals; D004285:Dogs; D004926:Escherichia coli; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000138:chemical synthesis*; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; D014764:Viral Proteins