High yield fermentation and purification of Tendamistat disulphide analogues secreted by Streptomyces lividans

Abstract

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.

Native-like β-structure in a Trifluoroethanol-induced Partially Folded State of the All-β-sheet Protein Tendamistat

“…The disulfide-deficient mutants were constructed as described (Haas-Lauterbach et al, 1993). Protein concentration was determined by UV absorption measurements using an absorption coefficient of A 0,1% 1 cm = 1.61 (Vertesy et al, 1984).…”

The nuclear‐magnetic‐resonance solution structure of the mutant α‐amylase inhibitor [R19L]Tendamistat and comparison with wild‐type Tendamistat

“…The fuzzy halo observed upon interaction with [R19L]Tendamistat indicated that the mutant shows a different behavior, when compared to wild-type Tendamistat. In the preparative isolation by fermentation [16], the yields of [R19L]Tendamistat were in the range 0.5 gA.…”

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

“…The detailed cloning and purification procedure has been published elsewhere. 27 For the 15 N isotope labeling of TM C45A/C73A, the Streptomyces media was prepared as follows: minimal medium (for 50 ml), 12.5 ml 20% (w/v) glucose, 0.5 ml 20% (w/v) ( 15 NH 4 )SO 4 , 5 ml 300 mM sodium potassium phosphate buffer, 1.5 ml 1 M MgSO 4 , 50 µl trace element solution, 0.7-0.75 g Escherichia coliamino acid hydrolysate, prepared in the manner described below. The volume was adjusted to 50 ml with sterile distilled water.…”

“…TM C45A/C73A was isolated from culture fluids of Streptomyces lividans and purified according to the procedure reported by Haas-Lauterbach et al 27 Uniform 15 N labeling to a level of 99% was obtained by growing the bacteria in minimal medium containing ( 15 NH 4 )SO 4 and a 15 N-labeled amino acid hydrolysate, obtained by enzymatic hydrolysis of sonified E. coli. Since Streptomyces lividans produces tendamistat only in complex media at a substantial amount, the minimal media have to be supplemented by 15 N-labeled amino acids and proteins.…”