Design And Assembly Of Collagen-like Peptide Conjugates

Collagen is the most abundant protein in extracellular matrix and one of the most commonly used natural polymers in the field of biomaterials.A de novo approach to designing collagen matrices which can have a broad impact on the understanding of matrix biology and our capacity to construct safe and medically useful biomaterials.Since native collagen is difficult to modify and can engender pathogenic and immunological side effects,its application on tissue application is limited.The collagen-like peptides obtained by chemical synthesis and bacterial expression are clean and easy to modify.Therefore,in recent years,they have been used as an ideal substrate for the collagen nanomaterials.However,it is still challenging to design short peptides to form uniform nanostructures.In this study,we used two different methods of chemical synthesis and bacterial heterogene expression to obtain collagen-like peptides.It relies on multiple weak forces to obtain highly controllable nanomaterials which provides a new strategy for the development of novel collagen-like peptide nanomaterials.The specific contents are as follows:?1?Collagen-like hybrid peptides?POG?₆ which can not form collagen triple helices and?POG?₈ which can form collagen triple helices were synthesized by solid-phase synthesis.The collagen-like hybrid peptides AP₆ and AP₈ were modified with 4,4'-methylene bis?phenyl isocyanate?at the nitrogen ends of collagen-likepeptide?POG?₆ and?POG?₈,respectively.As a control,the 4,4'-methylene bis was replaced by the rigid 1,5-naphthalene diisocyanate to obtain AnP₆ and AnP₈.The assembly morphology characterized by electron microscopy showed that AP₆ and AP₈ formed nanofibers with width of about 12 nm and 16 nm,respectively.AnP₆ and AnP₈ have no nanostructures.?2?Collagen-like peptides modified with alkyne and DNA modified with azide were coupled to form collagen-like peptide-DNA conjugates A-DNA1 and DNA2-C through azide-alkynyl click reaction catalyzed by copper ions.The equimole of A-DNA1,DNA2-C and B were mixed for heterologous assembly in the neutral 10 mmol·L^-1 PB and 10 mmol·L^-1 TAE buffer.CD results showed that both the collagen-like part and the DNA part could be assembled to form heterotrimer.AMF results showed that nanofiber structures with width of about 10 nm and height of about 2-3 nm could be formed in both buffers.?3?The purpose of the allogenic expression method of E.coli is to prepare the collagen-like peptides with longer length and richer amino acid composition.The recombinant plasmids were heterogenously expressed in E.coli to obtain Scl2A,Scl2C and Scl2ABC.CD characterized Scl2A,Scl2C and Scl2ABC with stable collagen triple helices and T_m values were 41.4?,46.3?and 40.3?,respectively.High stability collagen was obtained by expression which laid a foundation for further modification and induced assembly.