The identification, cloning, sequencing and expression in Escherichia coli of the gene encoding the Rt41A proteinase
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. Oligonucleotide primers were used in a PCR reaction to amplify a highly conserved region within the gene encoding an extracellular proteinase from Thermus sp. strain Rt41A. The PCR product was used as an internal gene probe in Southern hybridisations with genomic DNA from the same organism. Two genomic fragments which hybridised to the probe were cloned into Escherichia coli and sequenced. The sequence data showed that the two fragments overlapped and contained a 1233 bp open reading frame (ORFB) which had 71% identity to the sequence of Aqualysin I, an extracellular serine protease from Thermus aquaticus. A Shine-Dalgarno sequence and a putative 24 bp stem-loop structure were identified upstream from the start of ORFB. The end of ORFB was marked by two termination signals. The deduced peptide sequence of ORFB was 411 amino acids long and encoded a protein of 42.3 kDa. The structure of the deduced peptide was predicted to be a signal sequence (19 amino acids) followed by a pro-region (113 amino acids) and a catalytic domain (279 amino acids). The catalytic domain contained a catalytic triad of Asp39, His72 and Ser224, a feature typical of the Subtilases. Comparison of the peptide sequence of the catalytic domain with the database showed that it had high homology to other subtilases including Aqualysin I, Exoprotease A and Proteinase K. The Rt41A proteinase gene was expressed as a protein fusion to glutathione-S-transferase (GST) in E. coli. The fusion resulted the production of the proteinase at a level similar to that seen in the native organism. The recombinant protein was heat activated to remove the GST and pre-pro-regions before partial characterisation of the protein was performed. It was found that the recombinant protein retained the characteristics of the enzyme from the native organism. Amino-terminal sequencing and western blotting were used to confirm the identity of the recombinant protein. Removal of the putative signal sequence from the fusion resulted in a 30% reduction in the time of heat-treatment required to activate the proteinase.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/1993