Streptomyces griseus protease A (SGPA), B (SGPB) and Trypsin (SGT) are the extracellular serine proteases from Streptomyces griseus. SGPA and SGPB are chymotrypsin-like protease and SGT is a trypsin-like protease. It is such an interesting feature that prepro-sequence of SGPA and SGPB quite differ from that of SGT. SGPA, SGPB and SGT are very homologous not only to one another, but also to mammalian serine proteases. Streptokinase (SK) is an extracellular protein from several strains of streptococci that functions a plasminogen activator, a fibrinolytic agent without its protease activity. It has been proposed that these serine family proteases were related to Streptokinase in amino acid homology and molecular evolution. In our previous report, the genes encoding S. griseus protease A (sprA), B (sprB), Trypsin (sprT) were cloned from S. griseus ATCC 10137. In this study, the nucleotide sequence of SGPA and SGPB genes was determined and characterized. And several vectors for expression and secretion were constructed to develop the appropriate expression system, and the wild and mutated types of SGPA, SGPB, SGT and SK genes were expressed in E. coli, Bacillus subtilis and Streptomyces lividans. In addition the role of the propeptide of the microbial serine proteases was investigated.
The amino acid sequence deduced from the nucleotide sequence suggests that S. griseus protease A and B are produced as a precursor consisting of three portions. In the case of SGPB, an amino-terminal pre-sequence (38 amino acid residues), a pro-sequence (76 amino acid residues), and mature protease (185 amino acid residues). The first expression vectors for sprA and sprB were constructed by subcloning the gene fragments into multi-cloning site of E. coli-S. lividans shuttle vector. S. lividans 1326 transformed with the recombinant plasmids showed clear zone activity around its colony on the skim milk-YD plate. When the protease activity from culture medium was assayed using artificial substrate, it appeared after five days. The expressed protein was estimated to be approximately 19 kDa in 15% SDS-PAGE and purified to near homogeneity by FPLC column chromatography using Mono-S ion exchange resin. On the other hand, the high-level expression vector for Streptokinase has been constructed in which SK gene (skc) was placed behind a tac promoter, and ompA signal sequence. Recombinant SK was expressed in high-level and almost secreted into the periplasmic and extracellular fractions. Also the gene fragments encoding N-terminal (1-174) and C-terminal (254-414) amino acid residues respectively were successfully expressed in E. coli using the same expression system.
To investigate the effect of the pro-sequence on expression and activation of the protease, recombinant proteins of SGPB with and without the pro-sequence were expressed in Bacillus subtilis and E. coli. When the gene encoding the pro-mature of the SGPB was fused to signal sequence of Subtilisin E under the control of aprE promoter in Bacillus expression vector, SGPB was successfully secreted into the medium with correct cleaved N-terminal amino acid of mature sequence. But the removal of the pro-sequence of SGPB was found not to detect the expression of the mature protease. When the pro-mature of SGPB was fused to maltose-biding protein and expressed in E. coli, the fused protein was produced as an inclusion body. As the insoluble protein was solubilized in 6 M guanidine chloride and refolded, the protease was considerably activated. However for the fused SGPB without the pro-sequence, protease activity did not appear in the refolding. These results show that the pro-sequence of SGPB plays an important role in folding and activation of the expressed proteases and the activation pattern of the precursor protease is self-catalytic processing. Site-directed mutagenesis using PCR was carried out to replace active site His 57 of SGPB with Gly involving Gln, Glu and Lys. When the mutated protease genes were subcloned into Bacillus secretion vector and expressed in Bacillus subtilis, the protease mutants were not secreted and their activity were lost. From the analytical results of nucleotide sequence of the sprT it was proposed that 32 amino acid residues of signal sequence (pre) and 4 amino acid residues of pro-sequence exist in front of the mature part of SGT. The pro-sequence of SGT have been considered too short in length to play a general role. To express the SGT in B. subtitles and to understand the role of pro-sequence in protein folding, we subcloned two types of SGT gene (pro-deleted sprT and pro-sprT) under the downstream of the promoter and signal sequence of aprE gene from the B. subtilis. While the expression of the pro-sprT led to the production of inactive enzyme in which the pro-sequence was probably not autoprocessed, that of the pro-deleted sprT showed its clear activity. This result indicates that the pro-sequence is not necessary for the production of active form and does not affect the folding of SGT. Also in vitro SGT itself could refold without the aid of pro-sequence. Thus we assume that the pro-sequence acts as a self-inhibitor to the enzyme and is processed by other peptidase.
