Peng Y Y, Stoichevska V, Howell L, Dumsday G J, Brodsky B, Werkmeister J A and Ramshaw J A M
: J Bioengineer & Biomedical Sci
Collagens have a characteristic triple-helical structure with a (Gly-Xaa-Yaa)n repeating sequence. Collagen has long been used in various medical products in a range of clinical applications after extraction from animals. But there is growing concern with the use of animal collagens due to potential disease transmission. Collagen-like sequences with Gly every third residue have been observed in various bacteria. These bacterial collagens lack hydroxyproline, yet can form stable triple-helices at 35-38 ░C and can be fabricated for biomedical applications. We have expressed recombinant collagen-like proteins based on Scl2 from S. Pyogenes. This sequence includes a registration domain (V-domain) and a collagen domain (CL). Alternative constructs including V-CL-CL and structures containing functional sequences from other collagens were also examined. Constructs were inserted into pColdIII vector and trial quantities prepared by expression in shaker bottles. For selected constructs, the recombinant collagen was produced in a 2 L bioreactor at 37 ░C using a defined medium and a fed-batch (high cell density) fermentation strategy. At appropriate cell densities, the temperature was reduced to 25 ░C (cold shock) and collagen expression was induced with 1 mM IPTG. The recombinant protein expression was typically greater than 4 g/L prior to purification. Expressed proteins were purified using IMAC HyperCel-Sepharose. The V-domain was removed from collagen domains by enzyme digestion and collagens purified on SuperdexTM200. A variety of cell compatibility, immunogenicity and functional assays were performed on different constructs. No significant antibody responses have been observed, even when adjuvant was used. The functional sequences inserted from other collagens retained their functions.
Dr John A.M. Ramshaw is a protein chemist with Ph.D. and D.Sc. degrees awarded from the University of Durham, UK, and he is a Fellow of the Australian Academy of Technology Sciences and Engineering. He is a Chief Research Scientist at CSIRO, Australia, where he specialises in the development and evaluation of biomedical materials, especially those based on collagens. He has held major research grants from Industry and Government and has been a consultant to various national and international companies in the field of collagen chemistry. He has published over 160 papers, with an H-index of 30, which have been cited more than 3200 times.
Journal of Bioengineering & Biomedical Science received 289 citations as per Google Scholar report