High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity.
The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.
Reddy
A
A
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, P.O. Box 509, Empire State Plaza, Albany, NY 12201-0509, USA.
Grimwood
B G
BG
Plummer
T H
TH
Tarentino
A L
AL
D000595:Amino Acid Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology; D014162:Transfection / Q000235:genetics
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase