Orthogonal Test Design for Optimization of the Expression of Thymosin α1 and Thymosin α1- iRGD Gene in Engineered E.coli BL21 Strain

Xingzhen Lao¹, Meng Liu¹, Fang Zhang² and Heng Zheng^1,

¹School of Life Sciences and Biotechnology, China Pharmaceutical University, Nanjing, Jiangsu, PR China

²School of Pharmacy, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu, PR China

Cite this paper:
Xingzhen Lao, Meng Liu, Fang Zhang and Heng Zheng. Orthogonal Test Design for Optimization of the Expression of Thymosin α1 and Thymosin α1- iRGD Gene in Engineered E.coli BL21 Strain. American Journal of Medical Sciences and Medicine. 2014; 2(1):1-6. doi: 10.12691/ajmsm-2-1-1

Abstract

In order to increase the expression of fusion protein of thymosin α1(Tα1) and thymosin α1-iRGD (Tα1-iRGD) by the engineered E. coli BL21 strain, containing pET32a-Trx-Tα1 and pET32a-Trx-Tα1-iRGD plasmid, respectively. The key parameters that influenced the expression of Tα1 and Tα1-iRGD were optimized by employing an orthogonal experiment [L₂₅(5)³], including OD_600nm before induction, lactose concentration and induction time, each with five levels. The intensity of target protein band was scanned as a quantitative measure method of the protein expression, after electrophoresis separation of total soluble protein on SDS-PAGE. For Tα1 fusion protein, the optimal conditions were OD_600nm=0.6 before induction, 2.5 mmol/L lactose concentration, 4 hours induction time. Target protein expression levels could be achieved 32.8% of the total soluble proteins. For Tα1-iRGD fusion protein, the optimal conditions were OD_600nm=0.8 before induction, 7.5mmol/L lactose concentration, 4 hours for induction. Under the condition, the amount of expressed protein could reach 33.8% of the total soluble proteins. Whereas before optimization, the expression level of Tα1 and Tα1-iRGD fusion proteins were 20.6% and 9.0% of the total soluble proteins, respectively. The orthogonal test was proved to be an effective method to optimize the expression of target proteins.

Keywords:
thymosin α1 thymosin-iRGD gene expression orthogonal experiment anticancer

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Figures

References:

[1]	Goldstein,A.L, Low,T.L., McAdoo,M., Thurman,G.B., Rossio,J., Lai,C.Y., Chang,D., Wang,S.S., Harvey,C., Ramel, A.H., and Meienhofer,J., “Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide,” Proc Natl Acad Sci USA, 74(2).725-729. Feb.1977.
[2]	Goldstein,A.L., Slater,F.D. and White,A., “Preparation, assay, and partial purification of a thymic lymphocytopoietic factor (thymosin),” Proc Natl Acad Sci USA,56(3).1010-1017.Sep.1966.
[3]	Goldstein,A.L., “Clinical applications of thymosin alpha-1,” Cancer Invest, 12(5).545-547. May.1994.
[4]	Uicickas,Y.M., Quesenberry,J.C.P., Guo,D., Wells,K., Shan,J., Sanders,L., Skovron, M.L., Iloeje,U., Caldwell,C. and Manos,M.M., “Incidence of hepatocellular carcinoma among individuals with hepatitis B virus infection identified using an automated data algorithm,” J Viral Hepatol, 15(1). 28-36. Jan. 2008.
[5]	Li,Y., Chen, H., Li,X., Zhou,W., He,M., Chiriva-Internati, M., Wachtel, M.S. and Frezza, E.E., “A new immunomodulatory therapy for severe sepsis: ulinastatin plus thymosin{alpha} 1,”J Intensive Care Med, 24(1).47-53. Jan-Feb.2009.
[6]	Ancell, C.D., Phipps, J. and Young,L., “Thymosin alpha-1,” Am J Health Syst Pharm, 58(10). 879-885 May.2002.
[7]	Qin, Y., Chen, F.D., Zhou, L., Gong, X.G. and Han, Q.F., “Proliferative and anti-proliferative effects of thymosin alpha 1 on cells are associated with manipulation of cellular ROS levels,” Chem Biol Interact, 180(3).383-388. Aug.2009.
[8]	Moody,T.W., “Thymosin alpha 1 as a chemo- preventive agent in lung and breast cancer,” Ann NY Acad Sci,1112.297-304. Sep.2007.
[9]	Moody,T.W., Tuthill,C., Badamchian.M. and Goldstein A.L., “Thymosin alpha1 inhibits mammary carcinogenesis in Fisher rats,” Peptides, 23(5).1011- 1014.May.2002.
[10]	Garaci,E., Pica,F., Sinibaldi-Vallebona,P., Pierimarchi, P., Mastino,A., Matteucci,C. and Rasi,G., “Thymosin alpha(1) in combination with cytokines and chemotherapy for the treatment of cancer,” Int Immunopharmacol, 3(8). 1145-1150. Aug.2003.
[11]	Sungarian,A., Cielo,D., Sampath,P., Bowling,N., Moskal,P., Wands,J.R. and de la Monte, S.M., “Potential role of thymosin-alpha1 adjuvant therapy for glioblastoma,” J Oncol, 2009.
[12]	Low, T.L., McClure, J.E., Naylor, P.H., Spangelo, B.L. and Goldstein, A.L., “Isolation of thymosin alpha 1 from thymosin fraction 5 of different species by high performance liquid chromatography,” J Chromatogr, 266.533-544.Aug.1983.
[13]	Mutchnick, M.G., Lindsay, K.L., Schiff, E.R., Cummings, G.D., Appelman, H.D., Peleman,R.R., Silva, M., Roach, K.C., Simmons, F., Milstein, S., Gordon, S.C. and Ehrinpreis, M.N., “Thymosin alpha1 treatment of chronic hepatitis B: results of aphase III multicentre, randomized, double-blind and placebo-controlled study,” J Viral Hepat, 6(5).397-403. Sep.1999.
[14]	Chen, Y., Wang, A., Zhao, L., Shen, G., Cui, L. and Tang, K., “Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits,” Biotechnol Appl Biochem, 52(Pt 4), 303-312. Apr. 2009.
[15]	Ni, Y., Shi, Z., Wang, D., Yao, M., Qiao, M. and Guo, P., “High expression of thymosin alpha1 by injecting recombinant PVX vector into the tomato fruit,” Sheng Wu Gong Cheng Xue Bao, 25(4).537-541.Apr. 2009.
[16]	Pasqualini, R., Arap, W. and McDonald, D.M., “Probing the structural and molecular diversity of tumor vasculature,” Trends Mol Med, 8(12). 563-571. Dec. 2002.
[17]	Zetter, B.R., “On target with tumor blood vessel markers,” Nat Biotechnol, 15(12).1243-1244. Nov. 1997.
[18]	Arap,W., Pasqualini,R. and Ruoslahti,E., “Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model,” Science,279(5349). 377-380. Jan.1998.
[19]	Zitzmann, S., Ehemann, V. and Schwab, M., “Arginine- glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo,” Cancer Res, 62(18).5139-5143. Sep.2002.
[20]	Desgrosellier, J.S. and Cheresh, D.A., “Integrins in cancer: biological implications and therapeutic opportunities,” Nat Rev Cancer, 10(1). 9-22. Jan.2010.
[21]	Sugahara, K.N., Teesalu, T., Karmali, P.P., Kotamraju, V.R., Agemy, L., Girard, O.M., Hanahan, D., Mattrey, R.F. and Ruoslahti, E., “Tissue-Penetrating Delivery of Compounds and Nanoparticles into Tumors,” Cancer Cell,16(6). 510-520.Dec. 2009.
[22]	Sugahara, K.N., Teesalu,T., Karmali, P.P., Kotamraju, V.R., Agemy, L., et al. “Coadministration of a Tumor- Penetrating Peptide Enhances the Efficacy of Cancer Drugs,” Science,328(5981).1031-1035. May. 2010.
[23]	Lao, X., Liu, M., Chen,J. and Zheng,H., “A tumor- penetrating Peptide modification enhances the antitumor activity of thymosin alpha 1,” PLoS One, 8(8):e72242.Aug.2013.
[24]	Romano, D., Molla,G., Pollegioni,L. and Marinelli,F., “Optimization of human D-amino acid oxidase expression in Escherichia coli,” Protein Expr Purif, 68(1).72-78. Nov.2009
[25]	Volontè, F., Marinelli, F., Gastaldo, L., Sacchi, S., Pilone, M.S., Pollegioni, L. and Molla,G., “Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli,” Protein Expr Purif, 61(2).131-137. Oct.2008.
[26]	Li,C.M., Guo,C.J., “Optimization of liquid culture medium for phellinus linteus mycelia by orthogonal test,” Food SCi, 29(5).311-314.Oct.2008.
[27]	Cai, D.J., Shu, Q., Xu, B.Q., Peng, L.M. and He, Y., “Orthogonal test design for optimization of the extraction of flavonid from the fructus gardenia,” Biomed Envion Sci, 24(6).688-693. Dec.2011.