Expression of thecolHGene EncodingClostridium histolyticumCollagenase inBacillus subtilisand Its Application to Enzyme Purification

Abstract

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

“…A ColH fraction containing the gelatinase, prepared from C. histolyticum cultures as described previously (10), was used as partially purified ColH. ColH purified from cultures of recombinant B. subtilis DB104 (16) …”

“…The resulting plasmid was introduced into B. subtilis DB104. The transformant was grown, and the culture supernatant was subjected to ammonium sulfate precipitation and gel filtration as described elsewhere (16). The fractions with Pz-peptidase activity after gel filtration were applied to a hydrophobic interaction column (Ether-Toyopearl, bed volume 14 ml; Toso, Tokyo, Japan), which was pre-equilibrated with 50 mM Tris-HCl (pH 7.5) containing ammonium sulfate (40% saturation).…”

“…Growth Conditions-Recombinant B. subtilis strains were grown as described previously (16). For screening recombinant plasmids, E. coli DH5␣ transformants were grown in Luria-Bertani medium supplemented with 100 g of ampicillin/ml.…”

“…The active fraction was dialyzed against 50 mM Tris-HCl (pH 8.5), and then applied to a MonoQ column. Chromatography was performed as described elsewhere (16) except that the pH was changed to 8.5. A recombinant N-terminal peptide containing only S1 was produced as follows. An oligonucleotide encoding the C-terminal peptide of S1 (Val 564 to Ser 683 ) followed by a stop codon was prepared by polymerase chain reaction using two primers (5Ј-GTGCCTTTTGTAGCTGATGA-3Ј and 5Ј-CCGCGGTTAGGAATCACCTTCGTTTGGTA-3Ј) and pCHC201 plasmid DNA.…”

“…Oligonucleotides, 5Ј-GCAAATAATGTGTATAATCATG-TCTAAATAATTC-3Ј and 5Ј-GTGACTGGTGAGGCCTCAACCAAGTC-3Ј, were used as a mutagenic primer and a selection primer, respectively. The mutant enzyme, designated as rColH(E416D), was produced using a shuttle vector, pAT19 (17) as described elsewhere (16).…”