Cloning and expression of the enzymatic region of Streptococcal hyaluronidase

Document Type: Original Article

Authors

1 Department of Microbiology, Science and Research Branch, Islamic Azad University, Arak Branch, Arak, Iran

2 Biology Department, Payame Noor University, Arak, Iran

3 Molecular and Medicine Research Center, Department of Microbiology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

Abstract

Objective(s): Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity.
Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein) plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied.
Results: Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair) was inserted correctly into the recombinant vector. The expressed protein (65 KDa) was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient’s sera.
Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

Keywords


1. Ikebe T, Endo M, Ueda Y, Okada K, Suzuki R, Minami T, et al. The genetic properties of Streptococcus pyogenes emm49 genotype strains recently emerged among severe invasive infections in Japan. Jpn J Infect Dis 2004; 57:187-188.

2. Morosini M, Canton R, Loza E, Del Campo R, Almaraz F, Baquero F. Streptococcus pyogenes isolates with characterized macrolide resistance mechanisms in Spain: in vitro activities of telithromycin and cethromycin. J Antimicrob Chemother 2003; 52:50.

3. Cunningham MW. Pathogenesis of group a strepto-coccal infections. Clin Microbiol Rev 2000; 139:470-511.

4. Akhtar M, Bhakuni V. Streptococcus pneumoniae hyaluronate lyase: An overview. Curr Sci 2004; 86:285-295.

5. Butler L, Rainger G, Nash G. A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods. Exp Cell Res 2009; 315:3433-3441.

6.  Starr C, Engleberg N. Role of hyaluronidase in sub-cutaneous spread and growth of group A streptococcus. Infect Immun 2006; 74:40-48.

7. Schulze C, Bittorf T, Walzel H, Kundt G, Bader R, Mittelmeier W. Experimental evaluation of hyaluronidase activity in combination with specific drugs applied in clinical techniques of interventional pain management and local anaesthesia. Pain Phys 2008; 11:877-883.

8. Stern R, Jedrzejas M. Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 2006; 106:818-839.

9. Stern R. Hyaluronan metabolism: a major paradox in cancer biology. Pathol Biol (Paris) 2005; 53:372-382.

10. Steer A, Vidmar S, Ritika R, Kado J, Batzloff M, Jenney

A, et al. Normal ranges of streptococcal antibody titers are similar whether streptococci are endemic to the setting or not. Clin Vaccine Immunol 2009; 16:172-175.

11.  Abtahi H, Moradkhani A and Pakzad I.Cloning and expression of the Streptococcus pyogenes hyaluronidase gene in Escherichia coli. Aust J of Basic App Sci 2012; 6:95-99.

12. Mahmoudi S, Abtahi H, Bahador H, Mosayebi G, Salmanian AH. Production of Recombinant Streptokinase in E. coli and Reactivity with Immunized

Mice. Pak J Biol Sci 2010; 13:380-384.

13.  Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 1990; 276:172-174.

 

14.  Molaee N, Abtahi H,  Mosayebi G. Expression of recombinant streptokinase from streptococcus pyogenes and its reaction with infected human and murine sera. Iran J Basic Med Sci 2013; 16:985-989.

15. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.

16. Farjadi V, Abtahi H, Zolfaghari, Safieh Soufian MR, Hasanzadeh L. Expression, purification and evaluation of antigenicity of cagA antigenic fragment of helicobacter pylori. Jundishapur J Microbiol 2013; 6:e7367.

17. Hasanzadeh L, Ghaznavi-Rad E, Soufian S, Farjadi V, Abtahi H. Expression and antigenic evaluation of vacA antigenic fragment of helicobacter pylori. Iran J Basic Med Sci 2013; 16:836-839.

18. Dorfman A. Mucopolysaccharidases.In:Holowick SP, Kaplan NO.edirors. Methods in Enzymology. Academic Press, N.Y;1995.p.166–173.

20. Hynes W, Dixon A, Walton S, Aridgides L. The extracellular hyaluronidase gene (hylA) of Streptococcus pyogenes. FEMS Microbiol Lett 2000; 184:109-112.

21.  Mahmoudi S, Abtahi H,  Bahador A, Mosayebi G, Salmanian AH , Teymuri M . Optimizing of nutrients for high level expression of recombinant streptokinase using pET32a expression system.J Clin Med 2012; 7:109- 114.