Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins

Abstract

Thioredoxin is a small redox protein with an active-site disulfide/dithiol. The protein from Escherichia coli has been well characterized. The genes encoding thioredoxin in E. coli and in the filamentous cyanobacterium Anabaena PCC 7119 have been cloned and sequenced. Anabaena thioredoxin exhibits 50% amino acid identity with the E. coli protein and interacts with E. coli enzymes. The genes encoding Anabaena and E. coli thioredoxin were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate hybrid genes, coding for two chimeric thioredoxins. These proteins are designated Anabaena-E. coli (A-E) thioredoxin for the construct with the Anabaena sequence from the N-terminus to the middle of the active site and the E. coli sequence to the C-terminus, and E. coli-Anabaena (E-A) for the opposite construct. The gene encoding the A-E thioredoxin complements all phenotypes of an E. coli thioredoxin-deficient strain, whereas the gene encoding E-A thioredoxin is only partially effective. Purified E-A thioredoxin exhibits a much lower catalytic efficiency with E. coli thioredoxin reductase and ribonucleotide reductase than either E. coli or Anabaena thioredoxin. In contrast, the A-E thioredoxin has a higher catalytic efficiency in these reactions than either parental protein. Reaction with antibodies to E. coli and Anabaena thioredoxins shows that the antigenic determinants for thioredoxin are located in the C-terminal part of the molecule and retain the native conformation in the hybrid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics

“…From these results, our conclusions resemble those of Lim et al (1988) in relation to the Trx-reductase and ribonucleotide-reductase activities with E. coli-Anabaena Trx hybrids. In spite of the changes introduced on the NH 2 -side of Trx f in the construction of the Trx m/f hybrid, the f-side of the latter keeps, or even improves, its structural characteristics for an efficient FBPase binding.…”

“…After PCR amplification and isolation by agarose gel electrophoresis, we obtained the recombinants which we named TfN, TfC, TmN and TmC, respectively. The hybrid Trx f/m was generated following the Lim et al (1988) protocol. After digestion of TfN with NcoI and AvaII, and of TmC with AvaII and BamHI, the purified DNA fragments were ligated to each other with T4DNA-ligase, and subcloned in the NcoI and BamHI sites of the expression vector pET-3d.…”

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120

“…Measurements were made in a 1-cm-path-length cell in a total volume of 1 ml. The excitation wavelength was 280 nm, and emission was recorded from 290 to 400 nm as described previously (28).…”

“…Polyclonal antibodies to Anabaena strain 7119 thioredoxin were raised in a rabbit as described previously (28). The antibodies were precipitated from serum with 40% saturated ammonium sulfate and dialyzed against Tris-buffered saline (150 mM NaCI).…”

“…An isolate of this phage has been designated T7tasl for thioredoxin-altered specificity and presumably has a mutation in the T7 gene 5 (27). The T7tasl mutant polymerase will also accept other foreign thioredoxins (28). However, strains containing the Anabaena strain 7120 thioredoxin will not support the growth of even the less demanding virus.…”