Expression of human parathyroid hormone in mammalian cells, Escherichia coli and Saccharomyces cerevisiae.
The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor alpha expression system containing the factor alpha-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.
Rokkones
E
E
Institute of Medical Biochemistry, University of Oslo, Norway.
Kareem
B N
BN
Olstad
O K
OK
Høgset
A
A
Schenstrøm
K
K
Hansson
L
L
Gautvik
K M
KM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D010281:Parathyroid Hormone; D011994:Recombinant Proteins