Construction of a synthetic messenger RNA encoding a membrane protein.

Abstract

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.

• Authors: J L Rubenstein, T G Chappell
• Journal: The Journal of cell biology (J Cell Biol) Vol. 96 Issue 5 Pg. 1464-9 (May 1983) ISSN: 0021-9525 [Print] United States
• PMID: 6341380 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• G protein, vesicular stomatitis virus
• Membrane Glycoproteins
• Membrane Proteins
• RNA, Messenger
• Viral Envelope Proteins
• Viral Proteins

Topics

• Animals
• Dogs
• Escherichia coli
• Membrane Glycoproteins
• Membrane Proteins (genetics)
• Protein Biosynthesis
• RNA, Messenger (chemical synthesis)
• Viral Envelope Proteins
• Viral Proteins (genetics)