Enzymic cleavage of fusion protein using immobilized urokinase covalently conjugated to glyoxyl-agarose.
We immobilized urokinase (UK) by covalent attachment to activated Sepharose 6B-CL through multi-point amine coupling and evaluated its performance in cleaving a fusion protein, which consisted of recombinant human growth hormone (hGH) and a fragment of glutathione S-transferase that was linked by a tetrapeptide of a UK-specific recognition sequence. Packing densities of aldehyde groups on the activated agarose surface could be controlled in a gel range of 7-60 micromol/ml aldehyde by the amount of glycidol used. The immobilization yield was nearly 100% at pH 10.5, and the specific activity of the immobilized UK was equivalent to about 80% of soluble UK under the assay conditions. The immobilized UK showed an improvement in pH and thermal stability, probably due to the structural rigidity imparted by multi-point linkages to the matrix. The cleavage rate by the immobilized UK was lower than that of the soluble enzyme but the side reaction of cryptic cleavage was significantly decreased, which might suggest that the enzyme's specificity was altered by the immobilization. Cleavage yield in the column packed with immobilized UK was dependent on the feed rate, and the yield was approx. 80% of that of the soluble UK. The monomeric hGH could be obtained by selectively precipitating the uncleaved fusion protein and the GST fragments at an acidic pH.
Suh
Chang Woo
CW
Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, 1271 Sa-dong, Ansan 425-791, South Korea.
Choi
Gang Sun
GS
Lee
Eun Kyu
EK
D000345:Affinity Labels / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry*; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D008567:Membranes, Artificial; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D000345:Affinity Labels; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; C047264:sepharose CL 6B; D012685:Sepharose; D005982:Glutathione Transferase; D014568:Urokinase-Type Plasminogen Activator