CLONING, PURIFICATION AND CHARACTERIZATION OF COLLAGEN-LIKE PROTEIN FROM METHYLOBACTERIA SP. 4-46 AS POTENTIAL HALAL BIOMATERIAL FOR WOUND HEALING AID

Abstract

Collagen is a powerful biomaterial that has been used for many centuries for its benefits. The unique structure of collagen makes it distinct from other proteins. Collagen possesses beneficial characteristics that can be utilized in various industries, especially in pharmaceutical and biomedical. The sources of collagen can be found mainly in animals. In Islam, animal-based ingredients are the most critical matter. Whether the animal is slaughtered according to Shariah, not slaughtered or prohibited, matters to the Muslims. Therefore, the issue with animal-based collagen would be the “halalness” of the ingredient/product. Not only that, animal-based collagen would also come with zoonosis risks. Therefore, these issues can be solved with collagen from non-pathogenic bacteria with a halal pipeline in mind. One of the non-pathogenic bacteria is Methylobacteria sp. 4-46, whose genome possesses a collagen domain, although it is a soil bacterial. In this study, the sequence of Methylobacteria sp. 4-46 that contains the collagen domain was cloned into the expression vector pColdII. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and ready for protein expression. Beforehand, to validate the success of the cloning, colony PCR was done with the transformed E. coli and the DNA sequence was done. The sequence generated was 100% matched with the sequence in the NCBI database. To obtain the highest yield of collagen-like protein (CLP), the medium components and fermentation parameters were optimized. For M9 minimal medium components, Plackett-Burman design was used as preliminary screening, and KH2PO4, NH4Cl and CaCl2 were the most significant medium components, and following this, OFAT experiments were carried out. The ranges of the concentrations were obtained, and FCCCD was used to further optimized those medium component concentrations. After the ANOVA (R2=0.7996) and validation of FCCCD model, the best concentrations for KH2PO4, NH4Cl and CaCl2 were 6 g/L, 3 g/L and 250 mg/L, respectively. Next, the fermentation parameters were optimized using Box-Behnken design. After the ANOVA (R2=0.9683) and validation of Box-Behnken model, the best settings for fermentation parameters were 15 ?, 250 rpm and 0.8 mM for induction temperature, agitation rate and IPTG concentration, respectively. To obtain a high purity of recombinant CLP, FPLC with HisTrap column was used. The purification factor and yield of purified recombinant CLP were 3.07-fold and 67.76%, respectively. For the characterization of recombinant CLP, the CLP was identified using the western blot method and Orbitrap LC-MS. The calculated size of recombinant CLP was 34.9 kDa and the western blot method gave ~35 kDa. While LC-MS gave the result of 33.4 kDa. The differences were minute and negligible, thus the recombinant CLP was successfully validated. The secondary structure of recombinant CLP was also determined using circular dichroism, and it was confirmed to possess triple helix structure. The recombinant CLP was also tested using scratch assay to determine the wound healing properties and compared with allantoin, the commercial wound healing aid. The result showed that 6.25% v/v recombinant CLP possessed wound healing properties with only 26.1% less wound closure than 0.1% v/v allantoin.