Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.
In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.
Morosoli
R
R
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Ville de Laval, Canada.
Durand
S
S
Moreau
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology / Q000235:genetics*; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D043325:Xylan Endo-1,3-beta-Xylosidase
D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase