N-Glycosidase-carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation.
A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM-EndoF1 and CBM-PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM-EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM-PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4 degrees C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.
Kwan
Emily M
EM
The Protein Engineering Network of Centres of Excellence, 750 Heritage Medical Research Centre, Edmonton, AB T6G 2S2, Canada.
Boraston
Alisdair B
AB
McLean
Bradley W
BW
Kilburn
Douglas G
DG
Warren
R Antony J
RA
D002846:Chromatography, Affinity; D045247:Chryseobacterium / Q000201:enzymology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology; D005170:Factor X / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006639:Histidine / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004800:Enzymes, Immobilized; D006023:Glycoproteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D005170:Factor X; D012260:Ribonucleases; C029600:ribonuclease B; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase