Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11.
BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20-40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA-GtfJ fusion could not be removed from the cell by 5 M LiCl wash.
Hung
Jacky
J
Centre for Molecular Biotechnology, QUT, G.P.O. Box 2434, 4001, Brisbane, Qld, Australia.
Rathsam
Catherine
C
Jacques
Nicholas A
NA
Giffard
Philip M
PM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003553:Cystine / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D018021:Lithium Chloride; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics*; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; C105995:BspA protein, bacteria; D005936:Glucans; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D003553:Cystine; D013395:Sucrose; D005964:Glucosyltransferases; D018021:Lithium Chloride; D005947:Glucose