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A Novel Induction Strategy Based on Temperature and pH Optimization to Improve the Yield of Recombinant Streptokinase in Escherichia coli

Saeed Azadi, Seyyed Kazem Sadjady, Roya Solaimanian, Arash Mahboubi




Streptokinase (SK) is used for thrombolytic therapy of acute myocardial infarction. Recombinant fermentation is the most common and cost-effective procedure in the production SK. Nutrients composition, as well as such fermentation variables as temperature and pH, can affect the production level of protein-based drugs expressed in E. coli. In the present study, optimized recombinant streptokinase (r-SK) production in E. coli in the form of inclusion body (IB), the effect of induction temperature and extracellular pH were evaluated.

Methods and Results:

To maximize the expression level of r-SK three different induction temperatures were used to obtain the optimal temperature for r-SK production. The effect of extracellular pH on the level of r-SK production was also studied and the optimal pH was determined. To this purpose, 8 fermentation processes were applied and followed by: Cell disruption, Isolation of IBs, determination of target protein (SDS-PAGE, Western-blot). The biological activity of r-SK obtained from optimal conditions was determined in comparison with reference standard using gel clot method.

The highest amount of expression level and IBs formation were performed by combining two temperatures: 42˚C for the first two hours of induction followed by 39˚C at pH 6. The expression level of r-SK under these conditions shows an 11.6% increase in comparison with the control.



Choosing appropriate cultivation conditions can increase IB formation and use its advantages to produce higher quantities of r-SK with appropriate biological activity.



Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology. 2014; 5:172.

Spadiut O, Capone S, Krainer F, Glieder A, Herwig C. Microbials for the production of monoclonal antibodies and antibody fragments. Trends in Biotechnology. 2014; 32(1):54-60.

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.

Baeshen M N, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Baeshen NA, Redwan EM. Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J. Microbiol. Biotechnol.25, 953–962 (2015).

Ferrer-Miralles N, Domingo-Espín J, Corchero JL, Vázquez E, Villaverde A. Microbial factories for recombinant pharmaceuticals. Microbial Cell Factories. 2009; 8:17.

Kamionka M. Engineering of Therapeutic Proteins Production in Escherichia coli. Current Pharmaceutical Biotechnology. 2011; 12(2):268-274.

Peternel Š, Komel R. Active Protein Aggregates Produced in Escherichia coli. International Journal of Molecular Sciences. 2011; 12(11):8275-8287.

Balagurunathan B, Ramchandra NS, Jayaraman G. Enhancement of stability of r-SK by intracellular expression and single step purification by hydrophobic interacellular chromatography. Biochem Eng J. 2008; 39:84-90.

Baig F, Fernando LP, Salazar MA, Powell RR, Bruce TF, Harcum SW. Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation. Biotechnology and bioengineering. 2014; 111(5):980-999.

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

Nahalka J, Vikartovska A, Hrabarova E. A cross-linked IB process for sialic acid synthesis. J Biotechnol. 2008; 134:146-153.

Doglia SM, Ami D, Natalello A, Gatti-Lafranconi P, Lotti M. Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies. Biotechnol J. 2008; 3:193-201.

Ventura S, Villaverde A. Protein quality in bacterial inclusion bodies. Trends Biotechnol. 2006; 24:179-185.

Alonso MM, Montalbán NG, Fruitós EG, Villaverde A. Learning about protein solubility from bacterial inclusion bodies. Microb Cell Fact. 2009; 8:4.

Kennedy M, Krouse D. Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biotechnol. 1999; 23:456-475.

Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer WD. Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol. 1995; 21: 59-65.

Seeger A, Schneppe B, McCarthy JEG, Deckwer WD, Rinas U. Comparison of temperature and isopropyl-β-d-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Enzyme Microb Technol. 1995;17:947-953.

Waterborg JH, Matthews HR. The Lowry method for protein quantitation. Methods Mol Biol. 1994; 32:1-4.

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

Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci. 1979; 76:4350-4.

Marsh NA, Gaffney PJ. The rapid fibrin plate-a method for plasminogen activator assay. Thromb Hemost. 1977; 38:545–51

Li RY, Cheng CY. Investigation of IB formation in recombinant Escherichia coli with a bioimaging system. J Biosci Bioeng. 2009; 107:512-515.

Middelberg AP. Preparative protein refolding. Trends Biotechnol. 2002; 20:437–443.

Schugerl K, Hubbuch J. Integrated bioprocesses. Curr Opin Microbiol. 2005; 8:294–300.

Banerjee A, Chisti Y, Banerjee UC. Streptokinase a clinically useful thrombolytic agent. Biotechnology Advances. 2004; 22:287–307.

Schoner RG, Ellis LF, Schoner BE. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Nat Biotechnol. 1985;3: 151–154.

Balagurunathan B, Jayaraman G. Theoretical and experimental investigation of chaperone effects on soluble recombinant proteins in Escherichia coli: effect of free DnaK level on temperature-induced r-SKproduction. Syst Synth Biol. 2008;2:1-2.

Betiku E. Molecular Chaperones involved in Heterologous Protein Folding in Escherichia coli. Biotechnol Mol Biol Rev. 2006; 1:66-75

Strandberg L, Enfors S. Factors Influencing IB Formation in the Production of a Fused Protein in Escherichia coli. Appl Environ Microbiol. 1991; 57:1669-1674.

Hickey EW, Hirshfield IN. Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium. Appl Environ Microbiol. 1990; 56:1038-1045.

DOI: https://doi.org/10.22037/ipa.v1i2.18551


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