title: Expression of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403 in methylotrophic yeast Pichia pastoris.
abstract: The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P. pastoris (GS115) using the methanol inducible AOX1 promoter with an alpha-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P. pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P. pastoris.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shirin
authors.initials: S
authors.lastname: Tarahomjoo
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Katakura
authors.forename: Suteaki
authors.initials: S
authors.lastname: Shioya
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology
chemical_list: D009238:N-Acetylmuramoyl-L-alanine Amidase