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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
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Abstract

Introduction:

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.

 

Conclusions:

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


References

References:

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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