High-level heterologous expression and secretion in Streptomyces lividans of two major antigenic proteins from Mycobacterium tuberculosis.
Two major antigens from Mycobacterium tuberculosis were produced by Streptomyces lividans as secreted extracellular proteins. An expression-secretion vector had been constructed that contained the promoter of xylanase A and the signal sequence of cellulase A. The latter contained two initiation codons preceded by a Shine-Dalgarno sequence plus eight nucleotides complementary to the 16S rRNA. The genes encoding the 38-kDa (Rv0934) and 19-kDa (Rv3763) proteins, respectively, were amplified by polymerase chain reaction and cloned into that vector. The recombinant proteins were then purified from the culture supernatants of the clones. The yields after purification were 80 mg/L for the 38-kDa protein and 200 mg/L for the 19-kDa protein. Sequence analysis of the N-terminal sequences showed a deletion of seven or eight amino acids for the 38-kDa protein, while in the 19-kDa protein 22 or 23 amino acids were lost, as compared with the respective wild-type proteins. However, the 19 kDa recombinant protein had the same N-terminal sequence as the one recovered from the M. tuberculosis culture supernatant. The high yields obtained for these two proteins demonstrated the potential of S. lividans as an alternative host for the production of recombinant proteins from M. tuberculosis. The culture conditions have yet to be worked out to minimize proteolytic degradation and to recover intact products.
Tremblay
Donald
D
Centre de recherche en microbiologie et biotechnologie, INRS-Institut Armand-Frappier, Université du Québec, Ville de Laval, Canada.
Lemay
Johanne
J
Gilbert
Michel
M
Chapdelaine
Yvan
Y
Dupont
Claude
C
Morosoli
Rolf
R
D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
C087864:19 kDa antigen, Mycobacterium; C075966:Ag38 antigen, Mycobacterium tuberculosis; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins