Bacterial collagen-like proteins (CLP) contain [G-X-Y]\(_{n}\) amino acid repeats and form collagen helix – a hallmark of mammalian collagen [1]. Bacterial CLP differ from the mammalian collagen in amino acid composition at X and Y positions, lack of post-translational modifications, and ability to form superstructures. Nevertheless, CLP offer many potential applications as alternatives to mammalian collagen. Moreover, comparing with animal collagens, bacterial CLP could be more easily engineered that makes them a promising candidates for unique biomaterial applications.
In this study, we focused on the GrtB putative collagen-like protein encoded by Granulicella tundricola MP5ACTX9T. G. tundricola is acidophilic bacterium, growing in environments with a pH range of 3.5 to 6.5 . Bioinformatic analysis of GrtB protein revealed that it contains [G-X-Y]\(_{n}\) repeat region followed by the C-terminal domain related to SGNH hydrolase superfamily. The predicted function of this domain is related to GDSL-lipase activity. To date, only one CLP with an enzymatic domain has been described, making this a novel target for investigation [2].
The aim of this study was to express several constructs of the GrtB protein for characterisation of this unique protein. Different constructs where design, including: 1) Full length protein fused to His-tag, 2) N-terminal domain of the protein, containing collagenous repeats, 3) C-terminal domains, containing lipase domain. To achieve this, genes encoding distinct protein variants were amplified by PCR and cloned into pCoofy or pColdI vectors by restriction-ligation cloning or by ligation independent cloning, respectively. Recombinant plasmids were then transferred into E. coli cells for expression.
In conclusion, the designed and expressed different GrtB protein constructs enable further investigation into the structural and functional properties of this unique putative collage-like protein. Moreover, the obtained results will provide substantial insights into the novel properties of bacterial collagen-like proteins with enzymatic domains, which could have potential applications in drug delivery, tissue engineering, and the development of bioactive materials.