Overexpression of Soluble Human Thymosin Alpha 1 in Escherichia coli
Pei-Fu CHEN,
Hong-Ying ZHANG,
Geng-Feng FU,
Gen-Xing XU,
Ya-Yi HOU,
Pei-Fu CHEN
Medical School, Nanjing University, Nanjing 210093, China;
Search for more papers by this authorHong-Ying ZHANG
School of Life Sciences, Nanjing University, Nanjing 210093, China
Search for more papers by this authorGeng-Feng FU
Medical School, Nanjing University, Nanjing 210093, China;
Search for more papers by this authorGen-Xing XU
School of Life Sciences, Nanjing University, Nanjing 210093, China
Search for more papers by this authorCorresponding Author
Ya-Yi HOU
Medical School, Nanjing University, Nanjing 210093, China;
*Corresponding author: Tel/Fax, 86-25-83686441; E-mail, [email protected]Search for more papers by this authorPei-Fu CHEN,
Hong-Ying ZHANG,
Geng-Feng FU,
Gen-Xing XU,
Ya-Yi HOU,
Pei-Fu CHEN
Medical School, Nanjing University, Nanjing 210093, China;
Search for more papers by this authorHong-Ying ZHANG
School of Life Sciences, Nanjing University, Nanjing 210093, China
Search for more papers by this authorGeng-Feng FU
Medical School, Nanjing University, Nanjing 210093, China;
Search for more papers by this authorGen-Xing XU
School of Life Sciences, Nanjing University, Nanjing 210093, China
Search for more papers by this authorCorresponding Author
Ya-Yi HOU
Medical School, Nanjing University, Nanjing 210093, China;
*Corresponding author: Tel/Fax, 86-25-83686441; E-mail, [email protected]Search for more papers by this authorAbstract
Abstract Synthesized gene of human thymosin alpha 1 (Tα1) was inserted into pET-28a, pET-9c, pThioHis B, pGEX-2T or pBV222 and then inductively expressed in strains of Escherichia coli. Among the five expression systems, the BL21/pET-28a system provides the highest expression level of fusion protein in a soluble form, which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting fusion protein purified through nickel affinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity by SDS-PAGE. These results indicate the potential of this expression system for high-throughput production of recombinant Tα1.
Edited by Ren-Bao GAN
References
- 1 Goldstein AL, Low TL, McAdoo M, McClure J, Thurman GB, Rossio J, Lai CY et al. Thymosin α1: Isolation and sequence analysis of an immunologically active thymic polypeptide. Proc Natl Acad Sci USA, 1977, 74: 725–729.
- 2 Low TLK, Thurman GB, McAdoo M, McClure J, Rossio JL, Naylor PH, Goldstein AL. The chemistry and biology of thymosin. I. Isolation and characterization and biological activities of thymosin α1 and polypeptide β1 from calf thymus. J Biol Chem, 1979, 254: 981–986.
- 3 Sarandeses CS, Covelo G, Díaz-Jullien C, Freire M. Prothymosin α is processed to thymosin α1 and thymosin α11 by a lysosomal asparaginyl endopeptidase. J Biol Chem, 2003, 278: 13286–13293.DOI: 10.1074/jbc.M213005200
- 4 Leichtling KD, Serrate SA, Sztein MB. Thymosin alpha 1 modulates the expression of high affinity interleukin-2 receptors on normal human lymphocytes. Int J Immunopharmacol, 1990, 12: 19–29.DOI: 10.1016/0192-0561(90)90064-T
- 5 Knutsen AP, Freeman JJ, Mueller KR, Roodman ST, Bouhasin JD. Thymosin-α1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model. Int J Immunopharmacol, 1999, 21: 15–26.DOI: 10.1016/S0192-0561(98)00060-5
- 6 Moshier JA, Mutchnick MG, Dosescu J, Holtz TK, Akkary S, Mahakala K, Merline JR et al. Thymosin-α1, but not interferon-α, specifically inhibits anchorage-independent growth of hepatitis B viral transfected HepG2 cells. Heptol, 1996, 25: 814–820.DOI: 10.1016/S0168-8278(96)80283-2
- 7 Bianco-Batlles D, Naylor CW, Moshier JA, Dosescu J, Naylor PH. Clonal growth inhibition as a bioassay for thymosin alpha 1: Inactivation of Tα1 by trifluroacetic acid. Cell Mol Biol, 2001, 47: 157–160.
- 8 Baumann CA, Badamchian M, Golstein AL. Thymosin α1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways. Mechanism of Ageing and Development, 1997, 94: 85–101.
- 9 Roy R, Singh SM, Shanker A. Mechanism of thymocyte apoptosis induced by serum of tumor-bearing host: The molecular events involved and their inhibition by thymosin α-1. Int J Immunopharmacol, 2000, 22: 309–321.DOI: 10.1016/S0192-0561(99)00087-9
- 10 Beuth J, Schierholz JM, Mayer G. Thymosin α1 application augments immune response and down-regulates tumor weight and organ colonization in BALB/c-mice. Cancer Lett, 2000, 159: 9–13.
- 11 Moody TW, Fagarasan M, Zia F, Cesnjaj M, Goldstein AL. Thymosin alpha 1 down-regulates the growth of human non-small cell lung cancer cells in vitro and in vivo. Cancer Res, 1993, 53: 5214–8.
- 12 Giuliani C, Napolitano G, Mastino A, di Vincenzo, S, D'Agostini C, Grelli S, Bucci I et al. Thymosin-α regulates MHC class I expression in FRTL-5 cells at transcriptional level. Eur J Immunol, 2000, 30: 778–786.DOI: 10.1002/1521-4141(200003)30:3<778::AID-IMMU778>3.3.CO;2-9
- 13 Tzehoval E, Sztein MB, Goldstein AL. Thymosins alpha 1 and beta 4 potentiate the antigen-presenting capacity of macrophages. Immunopharmacology, 1989, 18: 107–113.DOI: 10.1016/0162-3109(89)90063-5
- 14 Paul S, Sodhi A, Biswas SK. Activation of murine bone-marrow derived macrophages in vitro with thymosin-alpha-1 to tumoricidal state: A comparative study on normal and tumour-bearing hosts. Int J Immunopathol Pharmacol, 2000, 13: 129–140.
- 15 Shrivastava P, Singh SM, Singh N. Activation of tumor-associated macrophages by thymosin alpha 1. Int J Immunopathol Pharmacol, 2004, 17: 39–47.
- 16 Huang Y, Chen Z, Zhou C, Yao H, Li M, Xu C. The modulation of thymosin alpha 1 in the maturation, differentiation and function of murine bone marrow-derived dendritic cells in the absence or presence of tumor necrosis factor-α. Int Immunopharmacol, 2004, 4(4): 539–546.DOI: 10.1016/j.intimp.2004.02.008
- 17 Hadden JW. Thymic Endocrinology. Annals of the New York Academy of Sciences, 1998, 840: 352–358.
- 18 Ancell CD, Phipps J, Young L. Thymosin alpha 1. Am J Health Syst Pharm, 2001, 58: 879–885.
- 19 Billich A. Thymosin alpha 1. SciClone Pharmaceuticals. Current Opin Investig Drugs, 2002, 3: 698–707.
- 20 Moscarella S, Buzzelli G, Romanelli RG, Monti M, Giannini C, Careccia G et al. Interferon and thymosin combination therapy in naïve patients with chronic hepatitis C: Preliminary results. Liver, 1998, 18: 366–369.
- 21 Rasi G, Pierimarchi P, Sinibaldi-Vallebona P, Colella F, Garaci E. Combination therapy in the treatment of chronic viral hepatitis and prevention of hepatocellular carcinoma. Int J Immunopharmacol, 2003, 3: 1169–1176.DOI: 10.1016/S1567-5769(03)00012-2
- 22 Garaci E, Pica F, Sinibaldi-Vallebona P, Pierimarchi P, Mastino A, Matteucci C et al. Thymosin α1 in combination with cytokines and chemotherapy for the treatment of cancer. Int J Immunopharmacol, 2003, 3: 1145–1150.DOI: 10.1016/S1567-5769(03)00053-5
- 23 Garaci E, Pica F, Rasi G, Favalli C. Thymosin alpha 1 in the treatment of cancer: From basic research to clinical application. Int J Immunopharmacol, 2000, 22: 1067–1076.DOI: 10.1016/S0192-0561(00)00075-8
- 24 Hadden JW. Immunodeficiency and cancer: Prospects for correction. Int Immunopharmacol, 2003, 3: 1061–1071.DOI: 10.1016/S1567-5769(03)00060-2
- 25 Wetzel R, Heyneker HL, Goeddel DV, Jhurani P, Shapiro J, Crea R, Low TL et al. Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene. Biochemistry, 1980, 19: 6096–6104.
- 26 Shmelev VA, Bunina ZF, Kudriavtseva, TIu, Zinchenko, EV, Boldyreva EF, Korobko VG. Isolation of a group of hybrid proteins consisting of tumor necrosis factor alpha and thymosin alpha 1. Mol Gen Mikrobiol Virusol, 1995, ( 1): 9–14.
- 27 Xiu ZY, Yu Y, Chen CQ. Fusion expression of human thymosin alpha 1 in Escherichia coli. Sheng Wu Gong Cheng Xue Bao, 2002, 18: 541–545.
- 28 Zhang J, Qin Y, Ou-Yang Q, Xu H, Zhou KF, Liu RH, Lou SL. Construction of shuttle expression vector and expression of thymosin α1 in Synechococcus sp. PCC7942. Shi Yan Sheng Wu Xue Bao, 2003, 36: 1–4.
