Expression and purification of a synthetic human obese gene product.
Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores. We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli. The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium. The protein was readily purified to homogeneity from the culture medium using a two-step procedure.
Altmann
S W
SW
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304-1104, USA.
Timans
J C
JC
Rock
F L
FL
Bazan
J F
JF
Kastelein
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D020738:Leptin; D008969:Molecular Sequence Data; D009765:Obesity / Q000235:genetics*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D020738:Leptin; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide