Studies On Cloning Of The Collagenase Gene ColG From Clostridium Histolyticum And Expression Of Recombinant ColG In Pichia Pastoris

Microbial collagenase (EC 3.4.24.3.) is a metalloproteinase which degrades helical regions of native collagen into small fragments. The preferred cleavage site is -Gly in the sequence -Pro-Xaa-Gly-Pro-. Seven different polypeptides with collagenolytic activity have been demonstrated in a culture of Clostridium histolyticum, a pathogenic clostridium causing myonecrosis, and these have been divided into two groups on the basis of substrate specificity. The two classes are immunologically crossreactive but have significantly different sequences and different specificities such that their actions on collagen are complementary. Other variants have been isolated from Bacillus cereus, Empedobacter collagenolyticum, Pseudomonas marinoglutinosa, and species of Vibrio and Streptomyces.Clostridial collagenase has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. Furthermore, Clostridium histolyticum can grow well in simple media, producing fairly large amounts of enzyme in the culture medium. Because of these characteristics, this enzyme is widely used for biochemical and physiological studies, e.g., collagen depletion from vertebrate tissues, cleavage of collagen linkers, and therapeutic purposes. The difficulty in separating individual enzymes and the lot-to-lot variation of commercial collagenase preparations limit its practical use. Cloning, nucleotide sequence analysis, and application of this knowledge to develop recombinant DNA technology for the corresponding gene(s) would facilitate the purification of Clostridium histolyticum collagenase and also increase our understanding of this enzyme.In this study, we first successfully isolated and purified crude collagenase from Clostridium histolyticum cultured in an improved medium. Second, the gene for collagenase ColG was cloned. The recombinant collagenase ColG was then expressed in Pichia pastoris and detected by polymerase chain reaction (PCR) and 4-Phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPA) hydrolyzation.The contents of this paper are focused on the following parts:1 Isolation and purification of crude collagenase from Clostridium histolyticum cultured in 10L supplemented medium.The bacterium Clostridium histolyticum was cultivated under established anaerobic submerged conditions in a complex culture medium containing 10g beef extract, 10g yeast extract, 10g peptone and 20-30g gelatin per liter, at a temperature of 37℃and at pH 7.5 over a period of 22h. The medium was supplemented by adding K2HPO4/NaH2PO4 (0.063mol/L) and FeSO4·7H2O (2.8mg/100ml). Crude collagenase was isolated and purified from 10L fermentation medium by ammonium sulfate precipitation, ultrafiltration/diafiltration, and ion-exchange chromatography. Specifically, cells were removed by centrifugation at 4,500 rpm for 15 min at 4°C. Ammonium sulfate was added to 60% saturation, and the precipitate was collected by centrifugation at 12,000 rpm for 15 min at 4°C. The pellet was dissolved in distilled water, and then desalted by ultrafiltration/diafiltration. The desalted fraction was applied to an ion-exchange fast protein liquid chromatography column (MonoQ; bed volume, 1 ml; Pharmacia LKB Biotechnology, Uppsala, Sweden) which had been pre-equilibrated with 50 mM Tris-HCl (pH 7.5). Proteins were eluted with a 30-ml linear gradient of 0 to 0.8 M NaCl in 50 mM Tris-HCl (pH 7.5). Enzyme activity was monitored by determination of Pz-PLGPA-hydrolyzing activity, and the active fractions were combined.2 Expression of recombinant collagenase ColG as secreted proteins in Pichia pastoris2.1 Construction of expression vector pPICZαC-colG Construction of the recombinant expression vector was based on the pPICZαC plasmid. First, the DNA sequence containing colG was cloned into the vector pMD18-T by using the genomic DNA from Clostridium histolyticum as the template. Secondly, the insert containing cDNA encoding collagenase ColG was prepared via PCR according to the vector pMD18T-colG. The upstream primer contains the recognition sequence for XhoI and coding sequences for the KEX2 protease recognition sequence (Lys–Arg). The downstream primer contains the recognition sequence for XbaI. The nucleotide sequences of the cDNA insert and flanking sequence were verified by sequencing. Results of the DNA sequence analysis of the recombinant expression vector pPICZαC-colG demonstrated that the cDNA encoding collagenase ColG was correctly inserted into the pPICZαC vector and that the sequence was consistent with that logged in GenBank (accession no. D87215).2.2 Transformation of Pichia pastoris and selection of ColG-expressing coloniesThe linearized recombinant expression vectors were introduced into Pichia pastoris X-33 by electroporation using MicroPulserTM electroporation apparatus (Bio-Rad, Hercules, Calif.). The pPICZαC blank plasmids were also transformed as a negative control. Transformed cells were selected by growth on yeast extract peptone dextrose (YPD) agar plates containing zeocin. After the multicopy transformants appeared, twelve colonies were cultured in a shaken flask and the enzyme production was monitored by determination of Pz-PLGPA-hydrolyzing activity. Twelve colonies and one negative control were cultured for 168 h under induction by methanol at pH 6.0. In clone 6, levels of ColG increased gradually after induction and reached a maximum at 144 h, then began to decrease. The maximum level was 25,320U/L. In clone 13, which was transformed with the pPICZαC blank plasmid, the level of recombinant ColG was undetectable.In order to ascertain whether the cDNA encoding ColG was integrated into the Pichia pastoris host cell genome, a PCR was performed on the transformed yeast genomic DNA. Agarose gel electrophoresis of the PCR products showed that the cDNA encoding ColG was indeed integrated into all 12 clones transformed with ColG recombinant expression vector pPICZαC-colG. There were no visible bands in the control sample which had been transformed with pPICZαC blank plasmid.In conclusion, crude collagenase was isolated and purified from Clostridium histolyticum which was cultured in a supplemented medium. Then the gene for collagenase ColG was cloned according to Clostridium histolyticum genomic DNA. The collagenase ColG Pichia pastoris expression vector pPICZαC-colG is successfully constructed and ColG is expressed in Pichia pastoris. This research lays foundation for the further functional studying of recombinant collagenase ColG.