THIOREDOXIN FROM BACILLUS-ACIDOCALDARIUS - CHARACTERIZATION, HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI AND MOLECULAR MODELING

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purifi ed to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-de pendent reduction of bovine insulin disulphides. The protein has a mol ecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edma n degradation after cleavage with trypsin and cyanogen bromide. The se quences of known bacterial Trxs were aligned at the active site: BacTr x has an identity ranging from 45 to 53% with all sequences except tha t of the unusual Anabaena strain 7120 Trx (37% identity). The gene cod ing for BacTrx was isolated by a strategy based on PCR gene amplificat ion and cloned in a plasmid downstream of a lac-derived promoter seque nce; the recombinant clone was used as the expression vector for Esche richia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-D-t hiogalactoside; expressed BacTrx represents approx. 5% of the total cy tosolic protein. CD spectra and differential scanning calorimetry meas urements demonstrated that BacTrx is endowed with a higher conformatio nal heat stability than the Trx from E. coli. Nanogravimetry experimen ts showed a lower content of bound water in BacTrx than in E. coli Trx , and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constr ucted by a comparative molecular modelling technique, using E. coli Tr x and Anabaena strain 7120 Trx as reference proteins. Increased networ ks of ion-pairs and shorter loops emerged as major features of the Bac Trx structure compared with those of the template proteins. The findin gs are discussed in the light of the current knowledge about molecular determinants of protein stability.