Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone.
Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.
Forsberg
G
G
KabiGen, Stockholm, Sweden.
Brobjer
M
M
Holmgren
E
E
Bergdahl
K
K
Persson
P
P
Gautvik
K M
KM
Hartmanis
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; D013917:Thrombin