Comparative Characteristics of Biotechnological Approaches to Obtaining Recombinant Human Cytokines in Bacterial Expressing Systems


  • Tetiana Natochii Igor Sikorsky Kyiv Polytechnic Institute
  • Valentina Motronenko Igor Sikorsky Kyiv Polytechnic Institute



Cytokines, Bacterial producers, Bacterial expression systems, Recombinant proteins, Biosynthesis


Background. Cytokines provide a link between cells and play an important role in the innate and adaptive immune response modulation. As mediators, they are active in very small amounts and effect many biological processes: embryonic development, nonspecific and specific immune responses, changes in cognitive function, to name a few. Recombinant microorganisms are commonly used to produce cytokine preparations. Perspectives of therapeutic use of recombinant cytokines make it urgent to create new and improve existing technologies for their biosynthesis, isolation and purification.

Objective. The purpose of the paper is analysis and comparison of biotechnological approaches for the production of human recombinant cytokines, based on current literature, and identifying promising ways to improve the efficiency of their production technologies.

Methods. The analysis and systematization of modern scientific works devoted to the production of recombinant cytokines with determination of peculiarities of the course of their biosynthesis process and to carry out comparative characteristics of different producers.

Results. Most often, to obtain recombinant cytokines, Escherichia coli based expression systems are used, as they are the best-studied to allow high yield of the target product. Their biosynthesis, as a rule, is carried out by a method of periodic fermentation with nutrition, and to the composition of the nutrient medium can be added stimulants of biosynthesis of different origin. Bacterial producers have several disadvantages: the accumulation of the target protein is most often in the form of Taurus inclusions, and the question remains of contamination of such drugs with bacterial endotoxins. To negate these drawbacks, they resort to optimizing the procedures for isolating and purifying recombinant proteins.

Conclusions. One of the promising areas of current research on the production of recombinant cytokines is the various technologies for obtaining the target protein in soluble form. However, the complexity of this task lies in the lack of the ability to create a universal method and requires an individual approach, depending on the producer and the final product. Also promising are studies to increase the yield of the target protein by changing the culture conditions and composition of the nutrient medium, eliminating endotoxin infection, and finding alternative bacterial expression systems.


Dinarello CA. Historical review of cytokines. Eur J Immunol. 2011;37(1):34-45. DOI: 10.1002/eji.200737772

Zdravkovic N, Rosic M, Lutovac M, Zdravkovic V. Physiology and pathology of cytokine: Commercial production and medical use. In: Rezaei N, editor. Physiology and Pathology of Immunology. InTech; 2017. p. 33-53. DOI: 10.5772/intechopen.72200

El-Baky NA, Linjawi MH, Redwan EM. Auto-induction expression of human consensus interferon-alpha in Escherichia coli. BMC Biotechnol. 2015;15(14):1-10. DOI: 10.1186/s12896-015-0128-x

Кriachok IA, Tytorenko IB. Granulocyte colony stimulating factor use during anticancer therapy. Clinical Oncol. 2015;3(19):64-8.

Skrypnik KA, Kosorukov VS. Human granulocyte-colony stimulating factor as a new therapeutic agent in clinic. Rossiyskiy Bioterapevticheskiy Zhurnal. 2011;10(2):19-24.

Veldkamp C, Koplinski C, Jensen D, Peterson F, Smits K, Smith B, et al. Production of recombinant chemokines and validation of refolding. Methods Enzymol. 2016;570:539-65. DOI: 10.1016/bs.mie.2015.09.031

Tripathi N. Production and purification of recombinant proteins from Escherichia coli. ChemBioEng Rev. 2016;3(3):116-33. DOI: 10.1002/cben.201600002

Burgess-Brown N, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. Protein Expr Purif. 2008;59(1):94-102. DOI: 10.1016/j.pep.2008.01.008

Schmidt F. Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol. 2004;65(4):363-72. DOI: 10.1007/s00253-004-1656-9

Kim S, Jeong H, Kim E, Kim J, Lee S, Yoon S. Genomic and transcriptomic landscape of Escherichia coli BL21 (DE3). Nucleic Acids Res. 2017;45(9):5285-93. DOI: 10.1093/nar/gkx228

Nausch H, Huckauf J, Koslowski R, Meyer U, Broer I, Mikschofsky H. Recombinant production of human Interleukin 6 in Escherichia coli. PLoS ONE. 2013;8(1):5-10. DOI: 10.1371/journal.pone.0054933

Wyre C. Recombinant protein production in Escherichia coli: Optimisation of improved protocols [thesis]. Birmingham: University of Birmingham; 2014.

Rosano G, Ceccarelli E. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172. DOI: 10.3389/fmicb.2014.00172

Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, et al. Microscale to manufacturing scale-up of cell-free cytokine production − A new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011;108(7):1570-8. DOI: 10.1002/bit.23103

Toghraie FS, Sharifzadeh SM, Ramezani A, Maymand EM, Yazdanpanah-Samani M, Ghaderi A. Cloning and expression of recombinant human Interleukin-7 in Chinese Hamster Ovary (CHO) Cells. Rep Biochem Mol Biol. 2017;6(1):66-73.

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(1):1-10. DOI: 10.1186/s12934-015-0222-8

Basu A, Li X, Leong SS. Refolding of proteins from inclusion bodies: rational design and recipes. Appl Microbiol Biotechnol. 2011;92(2):241-51. DOI: 10.1007/s00253-011-3513-y

Goncharuk DA, Tkach EN, Zeynalov OA, inventor; Skiff Npk OOO, assignee. Expression system and method of producing non-modified recombinant proteins in Escherichia coli using It. Russia patent 2604796. 2016 Dec 16.

Devi N, Adivitiya, Khasa YP. A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture. Appl Microbiol Biotechnol. 2016;100(23):9979-94. DOI: 10.1007/s00253-016-7683-5

Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol. 2013;23(3):393-402. DOI: 10.1016/

Gupta SK, Shukla P. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol. 2016;36(6):1089-98. DOI: 10.3109/07388551.2015.1084264

Shamonov NA. Development of industrial technology for the production of pegylated forms of interferon alfa-2a and alpha-2b [dissertation]. Moscow: GosNIIgenetika; 2015.

Slavchenko IY. The influence of temperature on the yield soluble human alpha interferon in the system of recombinant proteins overproduction using bacteriophage lambda. Biopolym Cell. 2002;18(5):436-41. DOI: 10.7124/bc.000623

Ueda T, Akuta T, Kikuchi-Ueda T, Imaizumi K, Ono Y. Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide. Protein Expr Purif. 2016;120:99-105. DOI: 10.1016/j.pep.2015.12.015

Mamat U, Woodard RW, Wilke K, Souvignier C, Mead D, Steinmetz E, et al. Endotoxin-free protein production − ClearColi™ technology. 2012.

Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, Kohl TA, et al. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microb Cell Fac. 2015;14(81):1-15. DOI: 10.1186/s12934-015-0241-5

Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact. 2016;15(33):5. DOI: 10.1186/s12934-016-0437-3

Schwarz H, Schmittner M, Duschl A, Horejs-Hoeck J. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS One. 2014;9(12):1-15. DOI: 10.1371/journal.pone.0113840

Lutsenko TN, Galkin AYu. Substantiation of biotechnological approaches of producing Interleukin-7 recombinant human. Trudy BGTU. 2015;4(177):188-97.

Krause M, Neubauer A, Neubauer P. The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli. Microb Cell Fact. 2016;15(110):1-13. DOI: 10.1186/s12934-016-0513-8

Wang J, Chen J, Xu R, Xu Z. Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. coli K12 system. Prep Biochem Biotechnol. 2008;38(3):271-81. DOI: 10.1080/10826060802165089

Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, et al. Production of biopharmaceuticals in E. coli: Current scenario and future perspectives. Microbiol Biotechnol. 2015;27(7):953-62. DOI: 10.4014/jmb.1412.12079

Bis RL, Stauffer TM, Singh SM, Lavoie TB, Mallela KMG. High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a. Protein Expr Purif. 2014;99:138-46. DOI: 10.1016/j.pep.2014.04.010

Slavchenko IY, Boreyko EV, Vorobey NV. Influence of various inductor concentrations on the human alpha-2b interferon production in the bacteriophage T7 RNA polymerase-base expression system in Escherichia coli cells. Biopolym Cell. 2003;5(19):457-62. DOI: 10.7124/bc.000675

Valdez-Cruz NA, Caspeta L, Pérez NO, Ramírez OT, Trujillo-Roldán MA. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb Cell Fact. 2010;9(18):1. DOI: 10.1186/1475-2859-9-18

Studier FW. New Studier media for auto-induction and other applications. Protein Expr Purif. 2005;41:207-34.

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):1. DOI: 10.1186/s12934-015-0222-8

Ouellette T, Destrau S, Ouellette T, Zhu J, Roach JM, Coffman JD, et al. Production and purification of refolded recombinant human IL-7 from inclusion bodies. Protein Expr Purif. 2003;30(2):156-66. DOI: 10.1016/S1046-5928(03)00134-7

Tripathi NK. Production and purification of recombinant proteins from Escherichia coli. ChemBioEng Rev. 2016;3:116-33. DOI: 10.1002/cben.201600002

Wingfield PT, Palmer I, Liang SM. Folding and purification of insoluble (inclusion body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2014;78(6):1-30. DOI: 10.1002/0471140864.ps0605s78

Anselment BRK. Optimization and modeling of protein refolding conditions [dissertation]. Munich: Technical University of Munich; 2012.

Swietnicki W. Folding aggregated proteins into functionally active forms. Curr Opin Biotechnol. 2006;17(4):367-72. DOI: 10.1016/j.copbio.2006.05.011

Saraswat M, Musante L, Ravidá L, Shortt B, Byrne B, Holthofer H. Preparative purification of recombinant proteins: current status and future trends. Biomed Res Int. 2013;2013:1. DOI: 10.1155/2013/312709

Yuan XL, Li Y, Pan XH, Zhou M, Gao QY, Li MC. Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells. Mol Biol. 2016;50(3):466-73. DOI: 10.1134/s0026893316030134

Zaremba-Czogalla M, Stumpp C, Bonifacio E, Paul R. Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris. Protein Expr Purif. 2015;110:66-71. DOI: 10.1016/j.pep.2015.02.013

Luo D, Wen C, Zhao R, Liu X, Liu X, Cui J, et al. High level expression and purification of recombinant proteins from Escherichia coli with AK-TAG. PLoS ONE. 2016;11(5):1-11. DOI: 10.1371/journal.pone.0156106

Thomson CA, Olson M, Jackson LM, Schrader JW. A simplified method for the efficient refolding and purification of recombinant human GM-CSF. PLoS ONE. 2012;7(11):1-6. DOI: 10.1371/journal.pone.0049891

Cook DP, Gysemans C, Mathieu C. Lactococcus lactis as a versatile vehicle for tolerogenic immunotherapy. Front Immunol. 2017;8(1961):1-16. DOI: 10.3389/fimmu.2017.01961

Ferrer-Miralles N, Villaverde A. Bacterial cell factories for recombinant protein production; expanding the catalogue. Microb Cell Fact. 2013;12(113):1-4. DOI: 10.1186/1475-2859-12-113

Zurita-Turk M, del Carmen S, Santos ACG, Pereira VB, Cara DC, Leclercq SY, et al. Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis. BMC Biotechnol. 2014;14(73):1-12. DOI: 10.1186/1472-6750-14-73

Song AA, In LLA, Lim SHE, Rahim RA. A review on Lactococcus lactis: from food to factory. Microb Cell Fact. 2017;16(55):1. DOI: 10.1186/s12934-017-0669-x

Aubry C, Michon C, Chain F, Chvatchenko Y, Goffin L, Zimmerli SC, et al. Protective effect of TSLP delivered at the gut mucosa level by recombinant lactic acid bacteria in DSS-induced colitis mouse model. Microb Cell Fact. 2015;14(176):1. DOI: 10.1186/s12934-015-0367-5

Cano-Garrido O, Rueda FL, Sànchez-García L, Ruiz-Ávila L, Bosser R, Villaverde A, et al. Expanding the recombinant protein quality in Lactococcus lactis. Microb Cell Fact. 2014;13(167):1. DOI: 10.1186/s12934-014-0167-3

Hanif MU, Gul R, Hanif MI, Hashmi AA. Heterologous secretory expression and characterization of dimerized bone morphogenetic protein 2 in Bacillus subtilis. Biomed Res Int. 2017;2017:1. DOI: 10.1155/2017/9350537

Westers L, Westers H, Quax WJ. Bacillus subtilis as cell factory for pharmaceutical proteins - a biotechnological approach to optimize the host organism. Mol Cell Res. 2004;1694(1):299-310. DOI: 10.1016/j.bbamcr.2004.02.011



How to Cite

Natochii, T., & Motronenko, V. (2019). Comparative Characteristics of Biotechnological Approaches to Obtaining Recombinant Human Cytokines in Bacterial Expressing Systems. Innovative Biosystems and Bioengineering, 3(3), 128–145.