Collagen Crosslinking Assisted By Incorporating Unnatural Amino Acid With Thioether Bond

Collagen triple helix,composed of three polypeptide chains,further forms higher-order proteins such as fibrillar collagen and network collagen by assembly and cross-linking.Collagen are highly desired biomaterials that are used extensively in tissue engineering and other fields.The researchers produced recombinant collagen by engineered microorganisms that avoids problems related to batch-to-batch variability,interspecies transmission of disease and so on.However,how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials.In this study,collagenous domains from Streptococcus pyogenes was taken as the research object.Unnatural amino acid(O-(2-bromoethyl)-tyrosine)was incorporated into collagen by two-plasmid expression system.The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation.It paves the way for the assembly of fibril.The main research contents and results are as follows:(1)The design strategy of incorporating O2beY into recombinant collagen and forming thioether bond to assist collagen cross-linking.Fragment B of collagen derived from S.pyogenes was used as the object.The unnatural amino acid O2beY was incorporated into its N-terminal,C-terminal,middle and two ends by genetic manipulation,which was expressed as V-NZ,V-CZ,V-MZ and V-2Z,respectively.Cysteine was designed and introduced at both ends of fragment B,which was expressed by V-2C.The recombinant collagen with O2beY and 2C was reacted in vitro,and the cross-linking of collagen was assisted by the thioether bond formed.This strategy was applied to the full-length region of the triple helix,and the V-SC12-2Z of O2beY and the V-SC12-2C of cysteine at both ends were designed respectively to further increase the molecular size of collagen solution and obtain larger-scale collagen fibers.(2)The heterologous expression of recombinant collagen V-NZ with O2beY.In order to obtain high purity V-NZ,the concentration of arabinose,induction temperature and time and the concentration of IPTG were optimized in the fermentation of recombinant collagen with O2beY by two-plasmid expression system.The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol·L-1 IPTG and 0.06%arabinose at 25? for 24 h,the yield is about 20.0 mg·L-1.Under these conditions,V-CZ,V-MZ and V-2Z were successfully heterologously expressed,with yields of 22.0,25.0 and 17.0 mg·L-1,respectively.(3)Cross-linking optimization and characterization of collagen mutants.In order to maintain the triple helix structure of collagen during cross-linking in vitro,the effect of cross-linking reaction system pH on the bonding efficiency was studied at 15?.The results showed that the formation rate of thioether bond was the highest when cross-linked in NH4HCO3 buffer with pH 9.0.The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with single O2beY and collagen molecule with cysteine mutation,which formed aggregates with the largest molecular size up to 1.0 ?m,but linear fibers could not be observed and gels could not be formed.The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with two O2beY and 2C,which formed aggregates with the largest molecular size up to 1.0?m,and a small amount of linear fibers can be observed,but the gels still could not be formed.(4)Heterologous expression and cross-linking characterization of collagen SC12 mutant.V-SC12,V-SC12-2Z and V-SC12-2C were successfully expressed in E.coli with yields of 58.0,45.0 and 50.0 mg·L-1,respectively.The mixed solution of V-SC12-2Z and V-SC12-2C with the concentration of 1.0 mmol·L-1 was gel at 20?.The aggregate with the largest molecular size of 1.8 ?m could be formed after cross-linking for 48 h,and the longer linear fiber could be formed after cross-linking for 7 d.