Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response.
The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.
Surek
B
B
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie und Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Federal Republic of Germany.
Wilhelm
M
M
Hillen
W
W
D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*
D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator