Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase.
A novel export-affinity fusion vector employing the gene encoding cyclomaltodextrin glucanotransferase (CGTase; cgt) from Bacillus circulans var. alkalophilus (ATCC 21783) is described. CGTase binds to various sugar polymers, which makes it simple to purify it to near homogeneity in a single step. The CGTase fusion protein vector was constructed by deleting the translational stop codons from the gene encoding CGTase (cgt) by in vitro mutagenesis. As models, genes encoding Escherichia coli alkaline phosphatase (APase; phoA) and Bacillus stearothermophilus (ATCC 12980) alpha-amylase (BStA; amy) were fused to cgt. Overexpression of wild type CGTase and the hybrid proteins under the control of the lac promoter caused a 'leaky phenotype' in E. coli, the outer membrane became permeable, which enabled the adsorption of the fusion proteins directly from the culture medium onto alpha-cyclodextrin (alpha-CD) coupled agarose. The hybrid proteins were eluted from the column with alpha-CD solution under mild conditions at pH 7.5. The CGTase-APase' fusion had a good in vivo stability, whereas the CGTase-BStA' was less stable. In the latter case, according to protein sequencing, the proteolytically sensitive site was on the BStA' side of the fusion. The C-terminus of CGTase was stable against proteolysis as shown by narrow pH range isoelectric focusing. The fused enzymes retained their biological activities.
Department of Biochemistry, University of Turku, Finland.
J
J
Hellman
P
P
Mäntsälä
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase