Using mutagenesis and molecular cloning techniques to identify key residues within the molecular recognition site of the Glucuronide Membrane Transporter (GusB).

Abstract

The glucuronide transport membrane protein (GusB) encoded by the gusB gene is essential for the survival of Escherichia Coli (E. coli) residing in the gut of vertebrate species. This gene, along with four others located on the GUS operon, allows E. coli to scavenge carbon from detoxification products produced by the host. These products are known as glucuronides, they are composed of two parts, one is a glycone (glucuronic acid), and the other is an aglycone (various molecules). GusB has shown varying affinity for the substrates that is transports therefore recognising the glycone part very specifically and the agylcone part indiscriminately. However, the binding mechanism used by this protein this is still unknown. Amino acid residues within GusB must be structurally relevant for molecular recognition of glucuronide molecules; however information relating this is not yet available. Therefore, identifying key residues within the molecular recognition site of GusB became the focus of this research project. Using E. coli as a model organism, site-directed mutagenesis and cloning techniques employed in the laboratory were used to create six residual amino acid changes within the Glucuronide Transporter Protein. These occurred at various positions within the predicted cytoplasmic region of GusB. By substituting amino acids to others with different charges and polarity, it was expected that protein folding mechanisms and the molecular recognition site would be disrupted. Glucuronide transport activity was predicted to change as a result. Successful cloning to create mutant GusB proteins could not be achieved however, sequence analysis of a previous plasmid pE349 encoding a mutant GusB found an unexpected amino acid mutation at position 218 of the glucuronide membrane transporter. Chromogenic GUS assays showed the change from Asparginine (uncharged) to Lysine (positively charged) caused a reduction in the transport rate of glucuronides. This led to questions as to whether this amino acid change was indeed part of the molecular recognition site. All steps undertaken for mutagenesis and cloning techniques have been detailed and proposals for further research are highlighted. It is hoped that by identifying key amino acid residues, this protein could be manipulated in the future to recognise very specific substrates for use in biosensor and report gene technology.