The cloning and characterization of an endoglucanase gene of Cellulomonas fimi
Creators: name: Wong, Wan Keung Raymond
Publisher: name: University of British Columbia
Year: 2010

description: Recombinant plasmid pEC2, which was formed by inserting a 5.2 kb BamHI fragment of C. fimi DNA into the BamHI site of pBR322, expresses endoglucanase activity in E. coli (55). Three deletion mutants of pEC2, designated pEC2.1, pEC2.2 and pEC2.3, contain inserts of C. fimi DNA of 1.9, 2.4 and 3.5 kb, respectively. pEC2 and the deletion mutants expressed the same level of endoglucanase activity. The' active polypeptide produced by the four plasmids had a Mr of 53,000. Restriction analyses showed that the activity was determined by a 1.4 kb fragment of the 5.2 kb C. fimi insert which was common to all four plasmids. This 1.4 kb fragment is contiguous with the N-termlnal coding region of the Tc[sup R] gene of pBR322. Promoter deletion and frameshlfting experiments showed that the Tc[sup R] promoter and translational signals were required for the expression of the endoglucanase activity. The activity was found in a fusion polypeptide (53 kDa) comprised of the N-terminus of the Tc[sup R] determinant and the endoglucanase, which was lacking its N-terminus. A partial BamHI digest of C. fimi DNA was used to isolate a new clone determining the same endoglucanase. As confirmed by Southern analysis, the new plasmid, pcEC2, contained an additional 0.8 kb of C. fimi DNA which was contiguous with the 5' end of the 5.2 kb insert of pEC2. The expression of endoglucanase activity by pcEC2 was dependent upon the Tc[sup R] promoter but was independent of the Tc[sup R] determinant translation signals. This indicated that the entire coding sequence of the endoglucanase gene was contained within a 2.2 kb (0.8+1.4) fragment of C. fimi DNA. The nucleotide sequences of the 0.8 and 1.4 kb DNA fragments were determined by sequencing overlapping deletions using the dideoxy termination method. The overlapping deletions of the 1.4 kb fragment were generated by the unidirectional ExoIII method, whereas those of the 0.8 kb component were created by a novel method. The complete gene sequence is 1347 bp long, with the first 226 bp contained in the 0.8 kb fragment and the remainder in the 1.4 kb fragment. The total G+C content of the gene is 72.5%. This high G+C content is reflected in a very restricted codon usage. The gene encodes a polypeptide of 449 amino acids, with the first 31 amino acids constituting a putative leader peptide which is not found at the N-terminus of the mature enzyme purified from C. fimi. This leader peptide appears to function in the export of the endoglucanase in E. coli. A strong compression of nucleotides was found during the sequencing of the non-coding strand within the region encoding this leader peptide, and subcloning procedures were used to resolve the compression. The calculated molecular weight of the predicted mature protein (418 amino acids) is 51,837. A striking proline-threonine sequence repeat of 23 residues occurs in the predicted amino acid sequence. This repeat is very similar to one found in the predicted amino acid sequence of a C. fimi exoglucanase (137). The functions of these repeated proline-threonine sequences are yet to be determined. Immunoadsorbent chromatography was used to purify the endoglucanase from E. coli. The endoglucanase gene was subcloned in Saccharomyces cerev islae and Rhodobacter capsulatus. In 5. cerevi siae, the endoglucanase gene was fused to the yeast preprotoxin gene; the encoded fusion polypeptide was secreted into the culture medium using the signal peptide of the killer toxin. In R. capsulatus, the endoglucanase gene was fused to the rxcA B870β gene; the encoded fusion polypeptide is presumed to be largely cytoplasmic.