Expression of a bacterial endo (1-4)-beta-glucanase gene in mammalian cells and post translational modification of the gene product.
An endo (1-4)-beta-glucanase gene C6.5 from Bacillus subtilis has been expressed in Chinese hamster ovary (CHO) cells and pancreatic 266-6 cells. The fusion gene, stably transfected into CHO cells consisted of the mouse Amy-2.2 signal peptide coding sequence and the endoglucanase gene C6.5 transcribed from the early SV40 promoter/enhancer, using the dihydrofolate reductase gene as a selective marker. The gene construct transfected into pancreatic 266-6 cells consisted of the mouse Amy-2.2 promoter/enhancer and signal peptide coding sequence and the same C6.5 sequences using the xanthine-guanine phosphoribosyl transferase gene (gpt) as the selective marker. The stably transfected CHO cells synthesized endoglucanase at 1.1 U/mg cell protein in a 72 h culture, with 89% of the activity secreted into the culture fluid in a glycosylated form of 66 kDa as compared with the unglycosylated 53 kDa form expressed in E. coli. Glycosylation did not change the specific activity, protease resistance, or cellulose binding of the endoglucanase as compared to the unglycosylated form of the enzyme from E. coli. The level of expression in the stably transfected pancreatic cells was substantially lower at 0.3 mU/mg cell protein with all detectable activity present in the culture fluid. The secreted enzyme from pancreatic cells was glycosylated with a mass similar to that secreted from CHO cells.
Zhang
J X
JX
Department of Microbiology, University of Guelph, Ontario, Canada.
Krell
P J
PJ
Phillips
J P
JP
Forsberg
C W
CW
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D051379:Mice; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D000516:alpha-Amylases / Q000235:genetics
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases; D002480:Cellulase