Cloning and expression of a cDNA for human thioredoxin

• Published in: The Journal of biological chemistry Vol. 263; no. 30; pp. 15506 - 15512
• Main Authors: Wollman, E E, d'Auriol, L, Rimsky, L, Shaw, A, Jacquot, J P, Wingfield, P, Graber, P, Dessarps, F, Robin, P, Galibert, F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.10.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; Dithiothreitol - pharmacology; DNA - analysis; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation; Genetic engineering; Genetic technics; Humans; Immunoglobulin M; Malate Dehydrogenase - metabolism; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular cloning; Molecular genetics; Molecular Sequence Data; NADP - metabolism; Recombinant Proteins - isolation & purification; RNA, Messenger - analysis; Thioredoxins - genetics; Human; Molecular hybridization; Purification; Messenger RNA; Complementary DNA; Thioredoxin reductase (NADPH); Transcription; Enzyme; Active site; Primary structure; Molecular cloning; Gene expression
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)37617-3

Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine). This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin. Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase. Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.