Synthesis of peptides as cloned ubiquitin extensions.
Oligonucleotides encoding four peptides ranging in length from 10 to 21 amino acids were cloned between the Afl2 and KpnI sites in the ubiquitin expression vector, pNMHUb. Escherichia coli AR13, a strain that contains a temperature-sensitive lambda repressor, was transformed by the plasmids, and upon shift to 42 degrees C, the cells produced large amounts of ubiquitin extended at its carboxyl terminus by each of the four peptides. Following a simple three-step purification of the ubiquitin fusion proteins, the peptides were cleaved from ubiquitin using a ubiquitin-alpha-protein hydrolase isolated from rabbit reticulocytes. The released ubiquitin molecules were shown to be competent in conjugation assays, and amino acid analyses of the purified peptides revealed that full length products were obtained in all cases. Since peptide yields varied from 2 to 4 mg/liter of culture medium, ubiquitin extensions provide an attractive alternative to solid phase synthesis for the production of peptides.
Yoo
Y
Y
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Rote
K
K
Rechsteiner
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012156:Reticulocytes / Q000201:enzymology; D014169:Transformation, Bacterial; D014452:Ubiquitins / Q000096:biosynthesis* / Q000235:genetics
D000596:Amino Acids; D009841:Oligonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D004262:DNA Restriction Enzymes