Thioredoxin, phage

Some bacteriophage encode their own DNA polymerases. However, they usually rely on the host cell to provide accessory proteins. The sequence of the DNA polymerase from phage T7 is closely homologous to that of the Klenow fragment and the 3D structures are similar. The 80-kDa T7 polymerase requires the 12-kDa thioredoxin from the host cell as an additional subunit. It has been genetically engineered to improve its usefulness in DNA sequencing 278... 

At the time of writing this chapter, three commercial random peptide display libraries are available. Two phage Ph.D. libraries (New England Biolabs) are constituted of random 7-mers and random 12-mers fused to pill protein of the filamentous phage Ml3. The FliTrx (Invitrogen) is an E. coli display library with constrained random dodeca-peptides inserted into flagellin-thioredoxin fusion protein [10]. 

Thioredoxin systems have also been demonstrated in L. leichmannii (39), rat Novikoff hepatoma (40), T4 phage infected E. coli (41, 42), yeast (43), rat liver (44), calf liver (45) and germinating wheat embryo (32). 

Both E. coli and T4 thioredoxin have recently been purified by immu-noabsorbent affinity chromatography. The specific immunoadsorbents were prepared by coupling the a-globular fractions of rabbit anti-thio-redoxin antisera to Sepharose. This technique makes it possible to isolate both thioredoxins from phage-infected cells by two consecutive chromatography steps on the specific adsorbents (125, 126). 

Of the thioredoxins studied thus far only the T4 phage thioredoxin is unable to serve as the reducing substrate for E. coli ribonucleotide... 

Thioredoxin (1,2) are small (M. 11-12,000) ubiquitous redox proteins with two half-cystine residues in the conserved active site structure Trp-Cys-Gly-Pro-Cys. The oxidized form Trx-S2 is reduced by NADPH and thioredoxin-reductase the reduced rorm Trx(SH)2 is a powerful protein disulfide oxido-reductase which regulatesthe activity of enzymes by thiol redox control it serves as hydrogen donor for various reductive enzymes such as ribonucleotide reductase or enzymes reducing sulfate or methionine sulfoxide. Also, Trx(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and assembly of filamentous phages (fl and M13), at least in E, ooti. 

Thioredoxin is required in E. QoVl for filamentous phage assembly. 

Both cyanobacterial thioredoxins were expressed in E. coli. One gram of bacteria yielded approximately 20 mg of thioredoxin. T-1 was able to restore the wild type phenotype in E. coli strains lacking thioredoxin except for the growth of T7 phage (2). T-2 was not effective in the... 

T7 DNA polymerase is a heterodimeric 92-kDa enzyme composed of the phage gene 5 protein and the host-encoded thioredoxin in a 1 1 stoichiometry. In addition to the highly processive polymerase activity, the native T7 DNA Pol has a strong... 

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