Coexpression of proteins with methionine aminopeptidase and/or N-myristoyltransferase in Escherichia coli to increase acylation and homogeneity of protein preparations.
New plasmid constructs described in this article allow the coexpression in bacteria of any protein with several different NMT proteins, including the recently cloned full-length human NMT1 and 2, and with increased expression of bacterial Met-AP. Through the use of these plasmids in different combinations it should be possible to improve the homogeneity of a large number of recombinant protein preparations by the complete removal of the initiating methionine and increased extent of N-myristoylation. The new reagents described in this article are available upon request.
Van Valkenburgh
Hillary A
HA
GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA.
Kahn
Richard A
RA
D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008322:Mammals; D063208:Methionyl Aminopeptidases; D019814:Myristic Acid / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D019814:Myristic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases