The complete gene sequence (sprE) for the glutamic-acid-specific serine protease (SGPE) of the gram-positive bacterium Streptomyces griseus is reported. The sprE gene encodes a 355 amino acid pre-pro-mature enzyme. The presence of a glutamic acid residue at the junction of the pro and mature segments of the protein suggests that the enzyme is self-processing. SGPE was found to have extensive homology with the S. griseus proteases A and B. However, there is an additional segment to the pro region of SGPE, lacking in proteases A and B, which has significant homology to the pro region of the alpha-lytic protease of the gram-negative bacterium Lysobacter enzymogenes. Expression of recombinant SGPE in Bacillus subtilis is also reported, and the enzyme is shown to be self-processing.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34 -40 (L2) and 67-70 (L3), a library was generated containing 2 ؋ 10 10 variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys 1 -Gln 9 were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K i values, but TM8, containing Ser 2 to Asp and Ser 4 to Ala substitutions, was the most selective having a nanomolar K i value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 M. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.Normal biological processes, including embryo implantation, developmental remodeling of the extracellular matrix, and wound healing, require active MMPs.5 Excess active MMPs can have pathological effects and are tightly regulated at the levels of transcription, zymogen activation, and inhibition by endogenous high affinity protein inhibitors, the TIMPs. Disruption of the balance between active MMPs and their inhibitors results in diseases linked to unregulated matrix turnover, including arthritis, cancer, cardiovascular diseases, nephritis, neurological disorders, tissue ulceration, and fibrosis (1-4). The mammalian TIMPs are a family of four two-domain proteins (TIMP-1-4), with an N-terminal domain of ϳ125 amino acids and a C-terminal domain of ϳ65 amino acids; each domain is stabilized by three disulfide bonds. They show 41-52% identity in pairwise sequence comparisons.In general, the TIMPs show little discrimination in their inhibition of the 23 human MMPs. TIMP-1 inhibits most MMPs but is an exceptionally weak inhibitor of MT1-MMP, MT3-MMP, and MMP-19 (1, 2). In contrast, TIMP-2 forms high affinity complexes with all MMPs with K i values in the nanomolar range (3). TIMP-3 has a more extended inhibitory range that includes several disintegrin-metalloproteinases (ADAMs) such as ADAM-10, ADAM-12, ADAM-17, ADAM-28, and ADAM-33 together with various ADAMTS disintegrin metal...

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.

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Streptomyces griseus protease B, a member of the chymotrypsin superfamily, is encoded by a gene that expresses a pre-pro-mature protein. During secretion the precursor protein is processed into a mature, fully folded protease. In this study, we constructed a family of genes which encode deletions at the amino-terminal end of the propeptide. The secretion of active protease B was seen to decrease in an exponential manner according to the length of the deletion. The results underscore the intimate relationship between folding and secretion in bacterial protease expression. They further suggest that the propeptide segment of the zymogen stabilizes the folding of the mature enzyme through many small binding interactions over the entire surface of the peptide rather than through a few specific contacts.