Proteinase K from Tritirachium album Limber. Characterization of the chromosomal gene and expression of the cDNA in Escherichia coli.
The cDNA and the chromosomal gene encoding proteinase K from Tritirachium album Limber have been cloned in Escherichia coli and the entire nucleotide sequences of the coding region, as well as 5'- and 3'-flanking regions have been determined. The deduced primary translation product consisting of 384 amino acid residues (molecular mass = 40,231 Da) contains an N-terminal region of 105 amino acids not present in the mature protein. By analogy to the evolutionary-related bacterial subtilisins and other serine proteinases it is inferred that the primary secreted product is a zymogen containing a 15-amino-acid signal sequence and a 90-amino-acid propeptide. The propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. The nucleotide-sequence analysis of the gene and its flanking regions has revealed that the proteinase-K gene is composed of two exons and one 63-bp-long intron located in the proregion. Furthermore, a putative promoter sequence and a capping site have been identified, suggesting that the transcription-start site is located 103-bp upstream of the ATG initiation codon. To express the proproteinase-K gene in E. coli, proproteinase-K cDNA was cloned in a plasmid vector under control of the tac promoter. The hybrid plasmid pSPPRO, constructed for this purpose, contained the cDNA coding for proproteinase K [from Ala (-91) to the C-terminal Ala (279)] fused to the N-terminal-signal-peptide sequence of the alkaline-phosphatase gene preceded by the tac promoter. E. coli BMH71-18, harbouring this plasmid, exhibited slight proteolytic activity when tested on skimmed-milk plates, suggesting that some fusion proteins were correctly secreted into the periplasm and processed to the mature proteinase K.
Gunkel
F A
FA
Institut für Biochemie, Technische Hochschule Darmstadt, Federal Republic of Germany.
Gassen
H G
HG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D015342:DNA Probes / Q000096:biosynthesis; D019286:Endopeptidase K; D004792:Enzyme Precursors / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D003904:Mitosporic Fungi / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D000596:Amino Acids; D015342:DNA Probes; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K