Refolding and purification of a urokinase plasminogen activator fragment by chromatography.
A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of Mr 10000-1500000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA.
Fahey
E M
EM
Department of Chemical Engineering, University of Bath, UK. cesjbc@bath.ac.uk
Chaudhuri
J B
JB
Binding
P
P
D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator