Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification.
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.
Jung
C M
CM
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Japan.
Matsushita
O
O
Katayama
S
S
Minami
J
J
Ohhira
I
I
Okabe
A
A
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D003094:Collagen; D017364:Collagenases