Improvement of solubility and yield of recombinant protein expression in E. coli using a two-step system

Tahereh Sadeghian-Rizi , Azade Ebrahimi, Fatemeh Moazzen, Hesam Yousefian, Ali Jahanian-Najafabadi

Abstract


Overexpression of recombinant proteins in Escherichia coli results in inclusion body formation, and consequently decreased production yield and increased production cost. Co-expression of chaperon systems accompanied by recombinant protein is a general method to increase the production yield. However, it has not been successful enough due to imposed intense stress to the host cells. The aim of this study was to balance the rate of protein production and the imposed cellular stresses using a two-step expression system. For this purpose, in the first step, green fluorescent protein (GFP) was expressed as a recombinant protein model under control of the T7-TetO artificial promoter-operator, accompanied by Dnak/J/GrpE chaperon system. Then, in the next step, TetR repressor was activated automatically under the control of the stress promoter ibpAB and suppressed the GFP production after accumulation of inclusion bodies. Thus in this step incorrect folded proteins and inclusion bodies are refolded causing increased yield and solubility of the recombinant protein and restarting GFP expression again. Total GFP, soluble and insoluble GFP fractions, were measured by Synergy H1 multiple reader. Results showed that expression yield and soluble/insoluble ratio of GFP have been increased 5 and 2.5 times using this system in comparison with the single step process, respectively. The efficiency of this system in increasing solubility and production yield of recombinant proteins was confirmed. The two-step system must be evaluated for expression of various proteins to further confirm its applicability in the field of recombinant protein production.


Keywords


Chaperons; E. coli; GFP; Inclusion bodies; Protein solubility; Recombinant proteins.

Full Text:

PDF

References


Ben-Nissan G, Vimer S, Warszawski S, Katz A, Yona M, Unger T, et al. Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun Biol. 2018;1(1):213-224.

Kyratsous CA, Silverstein SJ, DeLong CR, Panagiotidis CA. Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli.Gene. 2009;440(1-2):9-15.

Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv. 2012;30(5):1102-1107.

Panda AK. Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli. Adv Biochem Eng Biotechnol. 2003;85:43-93.

Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, et al. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One. 2011;6(7):e22981.

Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630-642.

Calloni G, Chen T, Schermann SM, Chang HC, Genevaux P, Agostini F, et al. DnaK functions as a central hub in the E. coli chaperone network.Cell Rep. 2012;1(3):251-264.

Aguilar-Rodríguez J, Sabater-Muñoz B, Montagud-Martínez R, Berlanga V, Alvarez-Ponce D, Wagner A, et al. The molecular chaperone DnaK is a source of mutational robustness. Genome Biol Evol. 2016;8(9):2979-2991.

Kedzierska S, Staniszewska M, Wegrzyn A, Taylor A. The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells. FEBS Lett. 1999;446 (2-3):331-337.

Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J. 2013;32(6):419-425.

Xiao S, Shiloach J, Betenbaugh MJ. Engineering cells to improve protein expression. Curr Opin Struct Biol. 2014;26:32-38.

de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007;7:32-40.

Dragosits M, Nicklas D, Tagkopoulos I. A synthetic biology approach to self-regulatory recombinant protein production in Escherichia coli. J Biol Eng. 2012;6(1):2-11.

Jahanian-Najafabadi A, Soleimani M, Azadmanesh K, Mostafavi E, Majidzadeh AK. Molecualr cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid. Res Pharm Sci. 2015;10(1): 84-89.

Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli. BMC Biotechnol. 2015;1(15):13-21.

Qi X, Sun Y, Xiong S. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb Cell Fact. 2015;14:24-35.

Garcia-Fruitos E. Inclusion bodies: a new concept. Microb Cell Fact. 2010;9:80-82.

Rodriguez-Carmona E, Cano-Garrido O, Seras-Franzoso J, Villaverde A, Garcia-Fruitos E. Isolation of cell-free bacterial inclusion bodies. Microb Cell Fact. 2010;9:71-79.

Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact. 2015;14:41-50.

Carrio MM, Villaverde A. Localization of chaperones DnaK and GroEL in bacterial inclusion bodies. J Bacteriol. 2005;187:3599-3601.

Chang CC, Song J, Tey BT, Ramanan RN. Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction. Brief Bioinform. 2014;15(6):953-962.

Rosano GL, Morales ES, Ceccarelli EA.New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 2019;28(8):1412-1422

Carrio MM, Cubarsi R, Villaverde A. Fine architecture of bacterial inclusion bodies. FEBS Lett. 2000;471(1):7-11.

Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172-188.

Salehinia J, Sadeghi HMM, Abedi D, Akbari V. Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies. Res Pharm Sci. 2018;13(6):566-574.

Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S. Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J. 2011;278(14):2419-2427.

Liu L, Yang H, Shin HD, Chen RR, Li J, Du G, et al. How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioengineered. 2013;4(4):212-223.

Martínez-Alonso M, González-Montalbán N, García-Fruitós E, Villaverde A. Learning about protein solubility from bacterial inclusion bodies. Microb Cell Fact. 2009;8(1):4-8.

Martínez-Alonso M, García-Fruitós E, Ferrer-Miralles N, Rinas U, Villaverde A. Side effects of chaperone gene co-expression in recombinant protein production. Microb Cell Fact. 2010;9(1): 64-69.

Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, et al. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact. 2008;7(1):11-28.


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.