| Abstract [eng] |
Collagen is a protein found in all animals. It is the main component of the extracellular matrix. A structural feature common to all collagens is a unique triple helix structure characterised by a repeating a.r. pattern (Gly – X a.r – Ya.r)n. However, over the last twenty years, the study of bacterial genomes has led to the identification of a large number of sequences with (Gly – X a.r. – Y a.r.)n repeats. Recombinant collagen-like proteins could replace mammalian collagens, the wider use of which has been hampered by batch-to-batch variability, widespread disease, allergic reactions in humans, and costly scale-up. Thermophilic bacteria could provide an alternative to mammalian collagens as they grow at higher temperatures and their collagen-like proteins could have thermal stability at higher temperatures. Previous work in the laboratory has shown that, among the bacterial strains present in the laboratory, the N-3 strain of Geobacillus lituanicus encodes a Bacillus anthracis BclB-like protein, named GclB. The BclB protein of B. anthracis forms a collagen-like structure – a triple helix. In the present work, the GclB protein of the N-3 strain of G. lituanicus is investigated in order to assess the expression of this protein in E. coli. In addition to the GclB gene, a glycosyltransferase gene, which is also expressed in B. anthracis, was cloned alongside the BclB protein. The expression of the glycosyltransferase gene next to GclB is thought to confer stability to this protein, which would lead to correct folding of this protein after expression. The results obtained showed that the glycosyltransferase gene and the GclB gene could be successfully cloned into the pET-Duet-1 vector. After cloning, expression was performed in E. coli BL21 (DE3) strain by inducing the cells with 0,1 mM IPTG at 37 °C for 4 hours. After induction, NDS-PAGE protein electrophoresis showed the glycosyltransferase gene in the gel, but no expression of the GclB gene. |
