A method for the high-level expression of a parathyroid hormone analog in Escherichia coli.
A recombinant parathyroid hormone analog, rPTH(1-34*), was obtained from Escherichia coli using a gene polymerization strategy. The PTH gene polymer contains up to 8 copies of the gene, each separated by a cleavable linker. The polymer was expressed at very high levels and formed inclusion bodies which could be easily isolated by low-speed centrifugation. A polyhistidine leader peptide allows rapid purification via nickel chelation chromatography of the PTH polymer solubilized from the inclusion bodies. Yields of greater than 500 mg/liter have been obtained. After isolating the polymer, monomeric rPTH(1-34*) is released from the polymer by chemical cleavage with cyanogen bromide. Following cyanogen bromide cleavage and high-performance liquid chromatography purification, highly purified, biologically active rPTH(1-34*) is obtained at a yield of approximately 300 mg/liter. This is a general strategy for the high-level production of a variety of peptides and small proteins.
Oldenburg
K R
KR
Affymax Research Institute, Palo Alto, California 94304.
D'Orfani
A L
AL
Selick
H E
HE
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D019379:Teriparatide; D014407:Tumor Cells, Cultured
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019379:Teriparatide; D000262:Adenylyl Cyclases