title: High-level expression of Aliciclobacillus acidocaldarius thioredoxin in Pichia pastoris and Bacillus subtilis.
abstract: Thioredoxins are ubiquitous proteins which catalyze the reduction of disulfide bridges on target proteins and are involved in many cellular reactions. In a previous work, a thioredoxin from the thermophilic organism Aliciclobacillus acidocaldarius (Alitrx) was purified, characterized, and its gene expressed in Escherichia coli. In order to produce larger quantities of Alitrx, the protein has been expressed in the methylotrophic yeast Pichia pastoris and in the gram positive bacteria Bacillus subtilis. The growth conditions of strains showing high-level expression of Alitrx were optimized for both systems in shake-flask cultures. Active proteins were secreted in the culture media at a level of approximately 0.9 and 0.5 g/l, respectively, for P. pastoris and B. subtilis. The proteins were purified almost to homogeneity by a thermal precipitation procedure, with a 90-fold and 50-fold higher total yield with respect to that obtained with the same protein expressed in E. coli. The results indicate that either of these two systems could be utilized as a host for large-scale production of recombinant Alitrx.
authors.affiliation: Institute of Protein Biochemistry, CNR, Via Marconi 10, 80125 Naples, Italy.
authors.forename: Filomena Anna
authors.initials: FA
authors.lastname: Digilio
authors.forename: Rosa
authors.initials: R
authors.lastname: Morra
authors.forename: Emilia
authors.initials: E
authors.lastname: Pedone
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bartolucci
authors.forename: Mosè
authors.initials: M
authors.lastname: Rossi
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins