Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 720285, 6 pages http://dx.doi.org/10.1155/2013/720285 Research Article Construction, Expression, and Characterization of Thymosin Alpha 1 Tandem Repeats in Escherichia coli Xiao-Chang Xue, Zhen Yan, Wei-Na Li, Meng Li, Xin Qin, Cun Zhang, Qiang Hao, Zeng-Lu Wang, Ning Zhao, Wei Zhang, and Ying-Qi Zhang State Key Laboratory of Cancer Biology, Department of Biopharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China Correspondence should be addressed to Ying-Qi Zhang; xue xiaochang@yahoo.com Received 11 June 2012; Revised 26 November 2012; Accepted 26 November 2012 Academic Editor: Fabio Ferreira Perazzo Copyright © 2013 Xiao-Chang Xue et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hymosin alpha 1 (T1), which is composed of 28 amino acids, has been commercialized worldwide for its immune-modulatory and antitumor efects. T1 can stimulate T cell proliferation and diferentiation from bone marrow stem cells, augment cell-mediated immune responses, and regulate homeostasis of immune system. In this study, we developed a novel strategy to produce T1 concatemer (T1C) in Escherichia coli and compared its activity with chemically synthesized T1. Results showed that T1C can more efectively stimulate T cell proliferation and signiicantly upregulate IL-2 receptor expression. We concluded that the expression system for T1 concatemer was constructed successfully, which could serve as an eicient tool for the production of large quantities of the active protein. 1. Introduction he tandem repeats of proteins and peptides are studied widely and formidable progress has been made in this ield. It was reported that tandem amino acid repeats have many functions of stabilizing proteins [1], maintaining conforma- tion [2], elevating activity, and increasing half-life of proteins in blood or tissues. Frasch and colleagues suggested that tandem repeats present in Trypanosoma cruzi transsialidase stabilized the catalytic activity. In addition, repeats present on T. cruzi shed proteins increased trans-sialidase half-life in blood from 7 to almost 35 h [3]. Some proteins that contain tandemly repeated sequences play important roles in cell membrane skeleton system [4, 5]. hymosin alpha1 (T1) is a heat-stable, acidic polypeptide composed of 28 amino acid residues blocked at the N- terminus by an acetyl group [6, 7]. It is an immune modiier that has been shown to trigger lymphocytes maturation, augment T cell function, induce T-cell diferentiation, and promote reconstitution of immune defects. All these indings showed that T1 could be a useful restorative therapeutic agent in the treatment of immunodeiciency diseases and immunosuppressed conditions [8–10]. In this study, T1C which was composed of three repeated copies of T1 was fuse-expressed with thioredoxin (trx) in E. coli TOP10 strain and puriied by heat treatment and Q-Sepharose Fast Flow ion-exchange chromatography. hen, T1C was released by treatment with 0.5 M Cyanogen bromide (CNBr) and puriied by SP-Sepharose Fast Flow chromatography. In our strategy, trx acts as a chaperon to help T1C folding and CNBr treatment removed any exogenous amino acid (such as Met at the N-terminus for translation start) from T1C molecule. So we can get the “natural” T1C. Finally, the biological activity of T1C on T lymphocyte proliferation and IL-2R expression was assessed. 2. Materials and Methods 2.1. Materials. Restriction enzymes, Taq DNA polymerase, and T4 DNA ligase were purchased from TaKaRa. Expression vector phioHisA and E. coli strain TOP10 (F-mcrAΔ(mrr- hsd RMS-mcrBC) 80 lacZΔM15 ΔlacX74 recA1 araΔ139Δ (ara-leu)7697 galU galK rpsL (Strr)endA1 nupG) were from Invitrogen. DNA fragments were synthesized in BIOASIA. Synthetic T1 (ZADAXIN) was from Sciclone Pharmaceuti- cals, USA. he anti-T1 antibody (ab55635) was purchased