Purification of collagen-like proteins of Lactobacillus fermentum 3872

Collagen-like proteins (CLPs) in bacteria have been attracting a lot of attention in recent years. They have important roles, in biofilm formation, bacterial adhesion and motility and immune evasion, these promoting pathogenesis in pathogenic bacteria. Their action in probiotic bacteria has not yet been studied. In many cases the function of CLPs in bacteria is still unknown. The most important aspect of their biology may be in connection with the potential uses of probiotics as novel antipathogenic agents at a time when resistance to conventional antimicrobials, particularly antibiotics, is becoming increasingly widespread, resulting in a public health emergency.

Genes for two CLPs, denoted clp1 and clp2 have been discovered in Lactobacillus fermentum 3872. Genes clp1 and clp2 were cloned into Escherichia coli for expression and purification of the proteins using a standard affinity chromatography methodology, IMAC, with a histidine tag added to the proteins. Three types of protein purification kits, with a Ni-NTA resin or nickel magnetic particles, were used in attempts to purify enough protein for downstream experiments, but this was not achieved. A partial purification, at low yield and with contamination, was made. Experiments are described showing expression and partial purification under varying conditions with the commercially available kits.

A conclusion from the study is that it is important to find a cloning method and competent cells that express the target proteins adequately. Once good expression is established, the expressing clones could be used for empirical testing of a variety of IMAC and other affinity purification methods to find a reliable method for generating high yields of pure protein. The purified proteins could then be applied in downstream experiments to understand the structure of the proteins and their biological functions in L. fermentum 3872.

The current state of knowledge of functions of CLPs in bacteria is reviewed, highlighting the approaches that have been used to study the proteins.