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Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

Year 2014, , 13 - 19, 01.03.2014
https://doi.org/10.5799/ahinjs.02.2014.01.0117

Abstract

Objective: Synthetic cationic antimicrobial peptide (SC-AMP) is an important and upcoming therapeutic molecule against conventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods: The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3) and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain) - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a concentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC) of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage.

References

  • Boman HG. Peptide antibiotics and their role in innate immu- nity. Annu Rev Immunol 1995;13:61-92.
  • Martin E, Ganz T, Lehrer RI. Defensins and other endogenous peptide antibiotics of vertebrates. J Leukoc Biol 1995;58:128- 136.
  • Sima P, Trebichavsky I, Sigler K. Mammalian antibiotic pep- tides. Folia Microbiol (Praha) 2003;48:123-137.
  • Noga EU, Silphaduang U. Piscidins: A novel family of peptide antibiotics from Wsh. Drug News Perspect 2003;16: 87-92.
  • Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710-720.
  • Devine DA, Hancock RE. Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 2002;8:703-714.
  • ZasloV M. Antimicrobial peptides of multicellular organisms. Nature 2002; 415:389-395.
  • Hancock RE, Rozek A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett 2002;206:143-149.
  • Andes D, Craig W, Nielsen A, Kristensen HH. In vivo phar- macodynamic characterization of a novel plectasin antibi- otic, NZ2114, in a murine infection model. Antimicrob Agents Chemother 2009; 53:3003-3009.
  • Carballar-Lejarazu R, Rodriguez MH, De La Cruz Hernan- dez-Hernandez F, et al. Recombinant scorpine: A multifunc- tional antimicrobial peptide with activity against different pathogens. Cell Mol Life Sci 2008;65:3081-3092.
  • Mygind PH, Fischer RL, Schnorr KM, et al. Plectasin is a pep- tide antibiotic with therapeutic potential from a saprophytic fungus. Nature 2005;437:975-980.
  • Nizet V, Ohtake T, Lauth X. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 2001;414:454-457.
  • Montecalvo MA. Ramoplanin: A novel antimicrobial agent with the potential to prevent vancomycin-resistant enterococ- cal infection in high-risk patients. J. Antimicrob. Chemother 2003;51:S31-S35.
  • Giles FJ, Redman R, Yazji S, Bellm L. Iseganan HCl: A novel antimicrobial agent, Expert Opin. Invest. Drugs 2002;11:1161- 1170.
  • Patra AK, Mukhopadhyay R, Mukhija R, et al. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 2000;18:182-192.
  • Surinder mohan singh, Amulya kumar panda. Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering 2005;99:303-310.
  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Foli Phenol reagent. J.Biol.Chem 1951;193:265.
  • Asoodeh A, Naderi Manesh H, Mirshahi M, Ranjbar B. Puri- fication and characterization of antimicrobial and antifungal and non haemolytic peptide from Rana Ridibunda. Journal of sciences, Islamic republic of Iran 2004;15:303-309.
  • Xu Z, Zhong Z, Huang L, et al. High level production of bio- active human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl. Microbiol. Biotechnol 2006;72:471- 479.
  • Katia Conceicao, Katsuhiro Konno, Michael Richardson, et al. Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedu- sa hypochondrialis. Peptides 2006;27:3092-3099.
  • Smith DB, Johnson KS. Single-step purification of polypep- tides expressed in Escherichia coli as fusion with gluthione S-transferase. Gene 1988;67:31-40
  • Chen YQ, Zhang SQ, Li BC, et al. Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fu- sion partners. Protein Expr Purif 2008;57:303-311.
  • Piers KL, Brown MH, Hancock RE. Recombinant DNA proce- dures for producing small antimicrobial cationic peptides in bacteria. Gene 1993;134:7- 13.
  • Zhang L, Falla T, Wu M, et al. Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria. Biochem Biophys Res Commun 1998;247:674-80.
  • Cao W, Zhou Y, Ma Y, et al. Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli. Protein Expr Purif 2005;40:404-410.
  • Lai YP, Peng YF, Zuo Y, et al. Functional and structural char- acterization of recombinant dermcidin-1L, a human antimi- crobial peptide. Biochem Biophys Res Commun 2005;328: 243-250.
  • Sanz L, Chen RQ, Pérez A, et al. cDNA cloning and func- tional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha- 1beta1 integrin. J Biol Chem 2005;280:40714-40722.
  • Chen X, Zhu F, Cao Y, Qiao S. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. Antimicrob Agents Chemother 2009; 53:3683-3689.
  • Bommarius B, Jenssen H, Elliott M, et al. Cost-effective ex- pression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 2010;31:1957-1965
  • Li JF, Zhang J, Song R, et al. Production of a cytotoxic cat- ionic antibacterial peptide in Escherichia coli using SUMO fusion partner. Appl Microbiol Biotechnol 2009;84:383-388.

Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

Year 2014, , 13 - 19, 01.03.2014
https://doi.org/10.5799/ahinjs.02.2014.01.0117

Abstract

Amaç: Sentetik katyonik antimikrobiyal peptid (SK-AMP) önemi gittikçe artan ve konvansiyonel antibiyotiklere alternatif olması beklenen terapötik bir moleküldür. Bu çalışmada SK-AMP’yi intein tabanlı bir sistem ile enzimatik parçalanma olmaksızın saflaştırmak amaçlanmıştır.Yöntemler: İntein sekansı pTYB11 vektöründen PZR kullanılarak amplifiye edildi ve inteinin N-terminal ucu SK-AMP ile bağlandı. Oluşturulan bu yapı soğuk şok ekspresyon vektörü MCS bölgesine klonlandı. pCOLDI ve rekombinan peptid kitin afinite kodonundan 50 mM DDT ile ayrılarak enzimatik ayrıştırma olmaksızın toplandı. Daha sonra toplanan peptitin miktarı belirlendi ve saflaştırılan pepitd yapının antimikrobiyal aktivitesi difüzyon yöntemi ile belirlendi.Bulgular: Başlangıçta intein bağlı SK-AMP, IPTG indüklenebilir E. coli BL21(DE3) ve tuz indüklenebilir E. coli GJ1158’de bir füzyon protein olarak eksprese edildi. Tuz ile indüklenen E. coli GJ1158’de kitin bağlayan kodonun kullanıldığı tek basamaklı saflaştırma ile 208 mg/L konsantrasyonunda çözünebilir SK-AMP elde edildi. Saflaştırılmış SK-AMP’nin antibakteriyel etkisi hem gram pozitif hem de gram negatif bakterilere karşı gösterildi ve minimum inhibitör konsantrasyonu belirlendi.Sonuçlar: İlk kez çözünebilir SK-AMP tuz ile indüklenebilir E. coli GJ1158’den kitin bağlayan kodon kullanılarak tek basamakta enzimatik ayrışma olmaksızın elde edildi

References

  • Boman HG. Peptide antibiotics and their role in innate immu- nity. Annu Rev Immunol 1995;13:61-92.
  • Martin E, Ganz T, Lehrer RI. Defensins and other endogenous peptide antibiotics of vertebrates. J Leukoc Biol 1995;58:128- 136.
  • Sima P, Trebichavsky I, Sigler K. Mammalian antibiotic pep- tides. Folia Microbiol (Praha) 2003;48:123-137.
  • Noga EU, Silphaduang U. Piscidins: A novel family of peptide antibiotics from Wsh. Drug News Perspect 2003;16: 87-92.
  • Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710-720.
  • Devine DA, Hancock RE. Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 2002;8:703-714.
  • ZasloV M. Antimicrobial peptides of multicellular organisms. Nature 2002; 415:389-395.
  • Hancock RE, Rozek A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett 2002;206:143-149.
  • Andes D, Craig W, Nielsen A, Kristensen HH. In vivo phar- macodynamic characterization of a novel plectasin antibi- otic, NZ2114, in a murine infection model. Antimicrob Agents Chemother 2009; 53:3003-3009.
  • Carballar-Lejarazu R, Rodriguez MH, De La Cruz Hernan- dez-Hernandez F, et al. Recombinant scorpine: A multifunc- tional antimicrobial peptide with activity against different pathogens. Cell Mol Life Sci 2008;65:3081-3092.
  • Mygind PH, Fischer RL, Schnorr KM, et al. Plectasin is a pep- tide antibiotic with therapeutic potential from a saprophytic fungus. Nature 2005;437:975-980.
  • Nizet V, Ohtake T, Lauth X. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 2001;414:454-457.
  • Montecalvo MA. Ramoplanin: A novel antimicrobial agent with the potential to prevent vancomycin-resistant enterococ- cal infection in high-risk patients. J. Antimicrob. Chemother 2003;51:S31-S35.
  • Giles FJ, Redman R, Yazji S, Bellm L. Iseganan HCl: A novel antimicrobial agent, Expert Opin. Invest. Drugs 2002;11:1161- 1170.
  • Patra AK, Mukhopadhyay R, Mukhija R, et al. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 2000;18:182-192.
  • Surinder mohan singh, Amulya kumar panda. Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering 2005;99:303-310.
  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Foli Phenol reagent. J.Biol.Chem 1951;193:265.
  • Asoodeh A, Naderi Manesh H, Mirshahi M, Ranjbar B. Puri- fication and characterization of antimicrobial and antifungal and non haemolytic peptide from Rana Ridibunda. Journal of sciences, Islamic republic of Iran 2004;15:303-309.
  • Xu Z, Zhong Z, Huang L, et al. High level production of bio- active human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl. Microbiol. Biotechnol 2006;72:471- 479.
  • Katia Conceicao, Katsuhiro Konno, Michael Richardson, et al. Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedu- sa hypochondrialis. Peptides 2006;27:3092-3099.
  • Smith DB, Johnson KS. Single-step purification of polypep- tides expressed in Escherichia coli as fusion with gluthione S-transferase. Gene 1988;67:31-40
  • Chen YQ, Zhang SQ, Li BC, et al. Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fu- sion partners. Protein Expr Purif 2008;57:303-311.
  • Piers KL, Brown MH, Hancock RE. Recombinant DNA proce- dures for producing small antimicrobial cationic peptides in bacteria. Gene 1993;134:7- 13.
  • Zhang L, Falla T, Wu M, et al. Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria. Biochem Biophys Res Commun 1998;247:674-80.
  • Cao W, Zhou Y, Ma Y, et al. Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli. Protein Expr Purif 2005;40:404-410.
  • Lai YP, Peng YF, Zuo Y, et al. Functional and structural char- acterization of recombinant dermcidin-1L, a human antimi- crobial peptide. Biochem Biophys Res Commun 2005;328: 243-250.
  • Sanz L, Chen RQ, Pérez A, et al. cDNA cloning and func- tional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha- 1beta1 integrin. J Biol Chem 2005;280:40714-40722.
  • Chen X, Zhu F, Cao Y, Qiao S. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. Antimicrob Agents Chemother 2009; 53:3683-3689.
  • Bommarius B, Jenssen H, Elliott M, et al. Cost-effective ex- pression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 2010;31:1957-1965
  • Li JF, Zhang J, Song R, et al. Production of a cytotoxic cat- ionic antibacterial peptide in Escherichia coli using SUMO fusion partner. Appl Microbiol Biotechnol 2009;84:383-388.
There are 30 citations in total.

Details

Primary Language English
Journal Section ART
Authors

K Seetha Ram This is me

M Mary Vijaya Kumar This is me

S Divya Sri This is me

N Bhargava Ramudu This is me

J B Peravali This is me

Kk Pulicherla This is me

Publication Date March 1, 2014
Published in Issue Year 2014

Cite

APA Ram, K. S., Kumar, M. M. V., Sri, S. D., Ramudu, N. B., et al. (2014). Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. Journal of Microbiology and Infectious Diseases, 4(01), 13-19. https://doi.org/10.5799/ahinjs.02.2014.01.0117
AMA Ram KS, Kumar MMV, Sri SD, Ramudu NB, Peravali JB, Pulicherla K. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. J Microbil Infect Dis. March 2014;4(01):13-19. doi:10.5799/ahinjs.02.2014.01.0117
Chicago Ram, K Seetha, M Mary Vijaya Kumar, S Divya Sri, N Bhargava Ramudu, J B Peravali, and Kk Pulicherla. “Cost Effective Purification of Intein Based Syntetic Cationic Antimicrobial Peptide Expressed in Cold Shock Expression System Using Salt Inducible E. Coli GJ1158”. Journal of Microbiology and Infectious Diseases 4, no. 01 (March 2014): 13-19. https://doi.org/10.5799/ahinjs.02.2014.01.0117.
EndNote Ram KS, Kumar MMV, Sri SD, Ramudu NB, Peravali JB, Pulicherla K (March 1, 2014) Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. Journal of Microbiology and Infectious Diseases 4 01 13–19.
IEEE K. S. Ram, M. M. V. Kumar, S. D. Sri, N. B. Ramudu, J. B. Peravali, and K. Pulicherla, “Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158”, J Microbil Infect Dis, vol. 4, no. 01, pp. 13–19, 2014, doi: 10.5799/ahinjs.02.2014.01.0117.
ISNAD Ram, K Seetha et al. “Cost Effective Purification of Intein Based Syntetic Cationic Antimicrobial Peptide Expressed in Cold Shock Expression System Using Salt Inducible E. Coli GJ1158”. Journal of Microbiology and Infectious Diseases 4/01 (March 2014), 13-19. https://doi.org/10.5799/ahinjs.02.2014.01.0117.
JAMA Ram KS, Kumar MMV, Sri SD, Ramudu NB, Peravali JB, Pulicherla K. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. J Microbil Infect Dis. 2014;4:13–19.
MLA Ram, K Seetha et al. “Cost Effective Purification of Intein Based Syntetic Cationic Antimicrobial Peptide Expressed in Cold Shock Expression System Using Salt Inducible E. Coli GJ1158”. Journal of Microbiology and Infectious Diseases, vol. 4, no. 01, 2014, pp. 13-19, doi:10.5799/ahinjs.02.2014.01.0117.
Vancouver Ram KS, Kumar MMV, Sri SD, Ramudu NB, Peravali JB, Pulicherla K. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. J Microbil Infect Dis. 2014;4(01):13-9.