Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99.
A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.
Kruse
S
S
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
Kleineidam
R G
RG
Roggentin
P
P
Schauer
R
R
D000626:Aminopeptidases / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant / Q000235:genetics; D003956:Dialysis; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009113:Muramidase / Q000378:metabolism; D009439:Neuraminidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016622:Silver Staining; D014466:Ultraviolet Rays
D017931:DNA Primers; D004274:DNA, Recombinant; D007527:Isoenzymes; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009113:Muramidase; D009439:Neuraminidase; D000626:Aminopeptidases; C070939:aminopeptidase K