Improved leader and putative terminator sequences for high-level production of Streptomyces subtilisin inhibitor in Escherichia coli.
A high-level production system in Escherichia coli for an alkaline serine protease inhibitor, termed Streptomyces subtilisin inhibitor (SSI), from S. albogriseolus S-3253 was established by replacing the SSI signal sequence with the OmpA signal sequence using the inducible pIN-III-ompA vector. Significant amounts of recombinant SSI, resulting from accurate cleavage of the OmpA signal peptide, were accumulated in the periplasmic space or excreted into the culture medium. The inhibitory activity of the processed protein against subtilisin BPN' was identical with that of authentic SSI. Furthermore, deletion of one of the putative dual terminators (terminator 1) resulted in a 1.9-fold increase in production. This effect on SSI gene expression efficiency was found to be governed mainly at the transcription level.
Taguchi
S
S
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Yoshida
Y
Y
Matsumoto
K
K
Momose
H
H
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013302:Streptomyces / Q000235:genetics*; D013381:Subtilisins / Q000037:antagonists & inhibitors*; D013728:Terminator Regions, Genetic / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins