Efficient Preparation Of Collagens Integrated With Different Functional Sites And Their Bioactivity Evaluation

Collagen is a unique,triple helical molecule which forms the major part of extracellular matrix.It is the most abundant protein in the human body,representing 30%of our dry weight.Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms,but are also signal molecules defining cellular shape and behavior.Collagens for biomedical applications are mainly obtained from animals,especially from bovines.This has led to an ongoing concern regarding the transmission of diseases,especially spongiform encephalopathies("mad cow disease").Bacterially derived triple-helical,collagen-like proteins are attractive as potential biomedical materials.The collagen-like domain of the Scl2 protein from S.pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triplehelix of animal collagens.It can,therefore,be considered as a structurally-stable "blank slate" into which various defined,biological sequences,derived from animal collagens,can be added by substitutions or insertions,to enable the production of novel designed materials to fit specific functional requirements.Firstly,in the present study,we used site directed mutagenesis to substitute two kinds of integrin binding functional sequences,one for RGD(Arg-Gly-Asp)and the other for GFPGER,into different locations of the triple-helical structure.In this case,we created two groups of collagens functionalized with different integrin sites.Our results showed that the expression level of recombinant Scl2 was 1.2 g/L in shaking flasks,when cells were grew in 2×YT medium.Similar to other collagens,the triple helical domains of bacterial collagens are particularly resistant to proteolysis.Then we developed a simple,scalable procedure using a combination of acid precipitation of the Escherichia coli host proteins and proteolysis of residual host proteins to produce purified collagens in large scale without the use of chromatographic methods.Pepsin digestion resulted in complete degradation of the endogenous proteins and the purified collagan-like proteins could be obtained.Five kinds of lyophilized powders of collagens were prepared using this method.Finally,the effectiveness of the integrin sites substituted into the modified collagens for enhancing cell binding was demonstrated using L929 cells,which has been known to possess the appropriate integrin receptor.When compared to the unmodified bacterial collagen,the modified collagens containing both RGD and GFPGER sites showed significant adhesion of L929 cells.Whereas,the modified collagens containing only RGD site inhibited the adhesion of L929 cells.These data showed that the bacterial collagen-like sequences can be modified to have different functions by inserting one or more unique biological domains.