Recombinant Expression Of Human Collagen Type ? Peptide Genes In Escherichia Coli

Collagens,widespread in animals,are a family of biological macromolecules that have a variety of members.Collagen type ?,as a member of collagen family,is the major component of hyaline cartilage,and collagen type ? and its polypeptides on cartilage-related diseases have a good therapeutic efficacy.However,the collagen extracted from animal tissues has some limitations in the development and utilization because of its inherent defects in performance and source.Fortunately,the recombinant human collagen can not only solve this problem,but also has some new characteristics.This paper mainly focused on human collagen type ?,found a potential functional peptide,and achieved expression in Escherichia coli,which would provide some suggestions for the development of collagen type ?-related products and have a more broad application trend.Initially,human collagen type ? gene COL2A1 was identified by bioinformatics,accession number NM₀33150.2,CDS 4257 bp,encoding 1418 amino acids.The COL2A1 polypeptide?134.4 kD,pI=8.74?was predicted by bioinformatics software,which has a signal peptide and was hydrophilic which could be used for recombinant expression of soluble protein.Moreover,a functional polypeptide in COLF1 domain called COLFc?24.9 kD,pI=7.69?was found,by predicting the secondary structure of COL2A1,which was a calcium-binding protein promoting the calcification of the cartilage matrix and playing an important role in the cartilage physiological,pathological calcification.At the same time,in order to promote the efficient expression of the exogenous genes in host bacteria,codon optimization of COL2A1 was carried out by using Gene tuner,and the new sequence was called COL2A1 opt.In addition,the COL2A1 and COL2A1 opt sequences were obtained by whole-gene synthesis,then the recombinant plasmids pCY2A1,pCY2A1 opt and pGSTCY2A1 opt were constructed successfully.The COLFc and COLFcopt sequences were cloned by PCR using pCY2A1 and pCY2A1 opt as templates,then,the recombinant plasmids pCYFc and pCYFcopt were successfully constructed by inserting into pET30 a.These recombinant plasmids were transformed into E.coli BL21?DE3?,BL21?DE3?pLysS and BL21?DE3?Rosetta competent cells respectively,constructing 15 gene engineering strains successfully.The recombinant protein expression level of DS-pCYFcopt was the highest after induction with 0.05 mM IPTG for 5 h.The result showed that the fused protein was recombinant COLFc with His-tag by Western Blot.Furtherly,the recombinant protein COLFc with molecular weight of about 32 kD was puried by nickel ion affinity chromatography and FPLC.