Correct processing and secretion of a human prostatic secretory protein (PSP94) in Escherichia coli.
The cDNA for PSP94, a cysteine-rich protein secreted by the human prostate, was unidirectionally digested with exonuclease III to generate deletion mutants with varying 5' ends. These were placed under the control of the lac promoter of the Bluescribe plasmid (pbs) to encode hybrid proteins containing the N terminus of beta-galactosidase (beta Gal) and various fragments of PSP94. Escherichia coli clones transformed by these constructs and expressing PSP94 epitopes were identified by radioimmunoassay of cellular and periplasmic extracts. One such clone (I-25) secreted most of its immunoreactive material into the periplasmic space. Nucleotide sequencing showed that a new consensus ribosome-binding site had been generated fortuitously, allowing expression of pre-PSP94 free of any beta Gal sequence. Periplasmic PSP94 is indistinguishable from the natural human protein, indicating correct processing and folding of this cysteine-rich protein in bacteria.
Linard
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CG
J.A. de Sève Laboratories of Biochemical and Molecular Neuroendocrinology, Clinical Research Institute of Montreal, Québec, Canada.
Mbikay
M
M
Benjannet
S
S
Lazure
C
C
Seidah
N G
NG
Chrétien
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D011467:Prostate / Q000378:metabolism*; D029584:Prostatic Secretory Proteins; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis
D002352:Carrier Proteins; D004274:DNA, Recombinant; D029584:Prostatic Secretory Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C028321:estramustine-binding protein