In vitro renaturation of recombinant human pro-urokinase expressed in Escherichia coli.
OBJECTIVE
Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli. It must be denatured and renatured in vitro so that it can acquire activity. This study aimed at increasing the renaturation yield of denaturant pro-urokinase.


METHODS
We evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol reagents. We also compared the effects of nonspecific additives, step-wise dilution and urea gradient dialysis.


RESULTS
We defined the optimal conditions of pro-urokinase renaturation with a yield of about 20%-30%.


CONCLUSIONS
Different recombinant denatured proteins have different renaturation conditions due to their different molecular sizes, molecular constructions, disulfide bond numbers, and hydrophobicity. The renaturation yield can be increased by optimizing the renaturation conditions of a specific protein.
Zhu
H
H
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China.
Liu
W
W
Shi
W
W
Xue
Y
Y
Kuai
L
L
Ma
Z
Z
D003545:Cysteine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012965:Sodium Chloride; D003553:Cystine; D014568:Urokinase-Type Plasminogen Activator; D005978:Glutathione; D003545:Cysteine; C067551:saruplase; D019803:Glutathione Disulfide