Earticle

초록

영어

Engineered synthetic cationic antimicrobial peptides are the potential alternative drugs to existing antibiotics. In the present study, a novel attempt for the intracellular production of engineered synthetic cationic antimicrobial peptide (escAMP) using Pichia pastoris was studied. The engineered synthetic cationic antimicrobial peptide gene was synthesized using overlapping PCR. An entirokinase and hydroxylamine hydrochloride cleavage sites are incorporated at N- and C- terminal end of escAMP respectively for easy purification. Later the gene was inserted into the MCS region of pPICZ-B vector. The synthetic peptide under the AOX1 promoter was integrated into the Pichia pastoris GS115 genome and the recombinant clones were screened by using antibiotic resistance. Expression profiles of recombinant peptide were done using glycerol and methanol based synthetic medium and analysed on 18 % Tricine-SDS-PAGE. Purification of the expressed peptide was done after cell disruption (10 cycles on time, 10 cycles off time and 10 min of total time) using 6X histidine tag followed by enzymatic cleavage. In this study, 67 gm of dry cell weight/L and 580 mg/L of purified escAMP was produced. The purified peptide is analysed for its anti microbial activity against different Gram positive and Gram negative microbes. For the first time smallest engineered synthetic cationic peptide was designed, cloned and expressed from methanol inducible Pichia pastoris GS115 and production ranges are encouraging.

목차

Abstract
 1. Introduction
 2. Materials & methods
  2.1. Strains and media
  2.2. Computational tools used
  2.3. PCR amplification
  2.4. Construction of recombinant vector
  2.5. Transformation to P. pastoris
  2.6. Expression of escAMP
  2.7. Purification of escAMP
  2.8. Antimicrobial activity assay
 3. Results & Discussion
  3.1. Synthesis of escAMP
  3.2. Construction of pPICZ-B-escAMP expression vector
  3.3. Expression of escAMP
  3.4. Purification of the peptide
  3.5. Antimicrobial activity assay
 4. Conclusion
 Acknowledgments
 References

키워드

저자

• Seetha Ram Kotra [ Department of Biotechnology, Acharya Nagarjuna University, Guntur – 522510, India ]
• K. Sobha [ Department of Biotechnology, R.V.R.& J.C. College of Engineering, Guntur – 522019, India ]
• V. Viharika [ Department of Biology, Bharathi Junior College, Chirala – 523155, India ]
• A. Pavan Kumar [ Department of Biotechnology, R.V.R.& J.C. College of Engineering, Guntur – 522019, India ]
• P. Vengala Rao [ Department of Biotechnology, Alagappa University, Karaikudi – 630003, India ]
• M. Mary Vijaya Kumari [ Department of Microbiology, M.V.R. P.G. College, Gajuwaka – 530026, India ]
• Kona Prasad [ Department of Biotechnology, R.V.R.& J.C. College of Engineering, Guntur – 522019, India ]
• G. Ravi Teja [ Department of Biotechnology, R.V.R.& J.C. College of Engineering, Guntur – 522019, India ]
• K. V. Rajesh [ Department of Biotechnology, Bapatla Engineering College, Bapatla – 522101, India ]
• JB Peravali [ Department of Biotechnology, Bapatla Engineering College, Bapatla – 522101, India ] Corresponding author

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  International Journal of Bio-Science and Bio-Technology

• 간기

  격월간

• pISSN

  2233-7849

• 수록기간

  2009~2016

• 등재여부

  SCOPUS

• 십진분류

  KDC 505 DDC 605