High yield fermentation and purification of Tendamistat disulphide analogues secreted by Streptomyces lividans.
In our studies of structure-function correlation of polypeptides we used Tendamistat (TM), an alpha-amylase-inhibitor of Streptomyces tendae, as a model to investigate the influence of different mutants on the expression and secretion of the protein. In addition, we examined the influence of replacing the two disulphide-bridges that stabilize the two-loop structure of the whole protein. The single mutants C27S, C27T, C45A, the double mutants C11A/C27A, C11A/C27S, C11A/C27T, C11A/C27L, C45/C73A and the fourfold mutant C11A/C27A/C45A/C73A were prepared. The mutated TM gene was expressed in S. lividans TK 24, which secretes the active form of the inhibitor into the culture medium. Compared with the wild-type, the double-mutated TM derivatives show an increase in secreted protein by a factor of two to ten. In contrast, the single-mutated inhibitor analogues show the reverse effect. In order to examine the influence of temperature and culture media on the production of protein derivative we used the most unstable C11A analogue. Our expression studies at 10, 19, 28 and 37 degrees C established 19 degrees C as the optimal temperature for production of the protein derivatives. The correlation between the stability and secretion of TM is discussed in the context of our knowledge of protein translocation in bacteria. Based on these experiments we optimized the fermentation parameters, isolated TM analogous on a large scale, and verified them.
Haas-Lauterbach
S
S
Institut für Organische Chemie, Johann Wolfgang Goethe Universität, Frankfurt am Main, Federal Republic of Germany.
Scharf
M
M
Sprunkel
B
B
Neeb
M
M
Koller
K P
KP
Engels
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D004269:DNA, Bacterial; D010455:Peptides; D000516:alpha-Amylases; C030526:tendamistate