Evaluation of a novel subtilisin inhibitor gene and mutant derivatives for the expression and secretion of mouse tumor necrosis factor alpha by Streptomyces lividans.
In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.
Lammertyn
E
E
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Belgium.
Van Mellaert
L
L
Schacht
S
S
Dillen
C
C
Sablon
E
E
Van Broekhoven
A
A
Anné
J
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D051379:Mice; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D015398:Signal Transduction; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D014178:Translocation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D001426:Bacterial Proteins; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C018150:subtilisin inhibitor protein, Streptomyces