Iranian Journal of Microbiology 2017. 9(2):64-73.

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli
Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph

Abstract


Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification.
Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting.
Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified.
Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis.


Keywords


Mycobacterium tuberculosis, Rv1733c, Cloning, Expression optimization, Protein purification

Full Text:

PDF

References


WHO Global Report. World Health Organization. Global tuberculosis report 2016. WHO/HTM/TB/2016.13. Geneva: World Health Organization; 2016.

Partnership WST. The global plan to stop TB 2011-2015: transforming the flight towards elimination of tuberculosis. Geneva: World Health Organization; 2010.

http://www.who.int/iris/handle/10665/44437

Lin MY, Geluk A, Smith SG, Stewart AL, Friggen AH, Franken KLMC, et al. Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination. Infect Immun 2007; 75:3523-3530.

Bertholet S, Ireton GC, Kahn M, Guderian J, Mohamath R, Stride N, et al. Identification of human T cell antigens for the development of vaccines against Mycobacterium tuberculosis. J Immunol 2008; 181:7948-7957.

Lew JM, Kapopoulou A, Jones LM, Cole ST. TubercuList-10 years after. Tuberculosis (Edinb) 2011; 91:1-7.

Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015; 10:845-858.

Larsen JE, Lund O, Nielsen M. Improved method for predicting linear B-cell epitopes. Immunome Res 2006; 2: 2.

Larsen MV, Lundegaard C, Lamberth K, Buus S, Lund O, Nielsen M. Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction. BMC Bioinformatics 2007; 8:424.

HLAPred: identification and prediction of HLA class I and class II binders,

http://crdd.osdd.net/raghava/hlapred/team.html

Doytchinova IA, Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 2007; 8:4.

Saha S, Raghava GPS. AlgPred: prediction of allergenic proteins and mapping of IgE epitopes. Nucleic Acids Res 2006; 34: W202-W209.

Sorensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 2005; 4:1.

Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 1996; 260:289-298.

Vincentelli R, Cimino A, Geerlof A, Kubo A, Satou Y, Cambillau C. High-throughput protein expression screening and purification in Escherichia coli. Methods 2011; 55:65-72.

Papaneophytou CP, Kontopidis G. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif 2014; 94:22-32.

Brennan WA, Lin SH (1996). Strategies for Protein Purification and Characterization, A Laboratory Manual. Cold Spring Harbor Press, NY, USA.

Green MR, Sambrook J (2012). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, USA.

Duong-Ly KC, Gabelli SB. Salting out of proteins using ammonium sulfate precipitation. Methods in Enzymol 2014; 541:85-94.

Laemmli UK. Cleaveage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685.

Zvi A, Ariel N, Fulkerson J, Sadoff JC, Shafferman A. Whole genome identification of Mycobacterium tuberculosis vaccine candidates by comprehensive data mining and bioinformatic analyses. BMC Med Genomics 2008; 1: 18.

Nguyen Thi LT, Sarmiento ME, Calero R, Camacho F, Reyes F, Hossain MM, et al. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection. Tubeculosis 2014; 94: 475-481.

Leyten EMS, Lin MY, Franken KLMC, Friggen AH, Prins C, van Meijgaarden KE, et al. Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis. Microb Infect 2006; 8: 2052-2060.

Schuck SD, Mueller H, Kunitz F, Neher A, Hoffmann H, Franken KLCM, et al. Identification of T-cell antigens specific for latent Mycobacterium tuberculosis infection. PLoS One 2009 ;4: e5590.

Black GF, Thiel BA, Ota MO, Parida SK, Adegbola R, Boom WH, et al. Immunogenicity of novel DosR regulon-encoded candidate antigens of Mycobacterium tuberculosis in three high-burden populations in Africa. Clin Vaccine Immunol 2009; 16: 1203-1212.

Broedel SE Jr, Papciak SM, Jones WR. The selection of optimum media formulations for improved expression of recombinant proteins in E. coli. Athena ES 2001; 2: 1-7.

Gekko K, Timasheff SN. Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry 1981; 20: 4667-4676.

Ramírez OT, Zamora R, Espinosa G, Merino E, Bolívar F, Quintero R. Kinetic study of penicillin acylase production by recombinant E. coli in batch cultures. Process Biochem 1994; 29: 197-206.


Refbacks

  • There are currently no refbacks.


Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.