Heterologous expression of full-length and truncated forms of the recombinant guluronate-specific alginate lyase of Klebsiella pneumoniae.
The alyA gene, which encodes a guluronate-specific alginate lyase from Klebsiella pneumoniae, was cloned into the plasmid pCT54 to facilitate heterologous expression of the enzyme in Escherichia coli. The greatest enzyme specific activity was observed when the complete gene and flanking regions of DNA (contained within a 1.95 kb HindIII fragment) were cloned in the correct orientation relative to the vector-encoded trp promoter. Heterologous expression of the intact gene complete with flanking regions resulted in a 20-fold increase in enzyme yield compared to the original construct, pRC5. PCR mutagenesis and/or restriction endonuclease digestion was used to generate a series of fragments containing either the whole or truncated versions of the gene. Active enzyme was produced from constructs in which the region encoding the signal peptide had been deleted, although the recombinant protein was retained within the bacterial cells. An internal methionine (position 74) could be used as a start site for translation but resulted in the accumulation of inactive protein within inclusion bodies.
Hicks
S J
SJ
School of Molecular and Medical Biosciences (Biochemistry Unit), University of Wales, Cardiff, Wales.
Gacesa
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D006603:Hexuronic Acids / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism / Q000648:ultrastructure; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D017931:DNA Primers; D006603:Hexuronic Acids; D011994:Recombinant Proteins; C007896:guluronic acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase