Earticle

초록

영어

The newly discovered Th2 pro-inflammatory cytokine, interleukin-17E belongs to the member of IL-17 family. In this study, bioactive recombinant mutated human IL–17E (rhIL–25) was synthesized using overlapping PCR strategy and amino acid mutations were carried out using site directed mutagenesis. Four cysteins at 78th, 83rd, 136th and 138th positions were involved in disulphide bond formation and were responsible for biological activity of mature protein. These four cysteins were replaced with serine using nucleotide substitution and the desired outcome was cloned into expression vector pRSET-A followed by expressed in a salt inducible Escherichia coli GJ1158. The transformants were selected by ampicillin resistance marker and also by DNA sequencing. SDS–PAGE analysis confirms 17.06 kDa purified protein against low molecular weight protein marker. Protein quantification was carried out using Lowry’s method. Approximately 104 mg/L of recombinant IL-17E was produced at 37 ℃. Biological activity of protein was determined by the release of IL–6 from PBMC cells using rhIL–17E. This is the first report on production of interleukin-17E structural variant with enhanced specific activity without compromising the biological activity.

목차

Abstract
 1. Introduction
 2. Material and Methods
  2.1. Microbial Strains and Plasmid
  2.2. Medium Composition
  2.3. Gene Amplification using PCR
  2.4. Site Directed Mutagenesis
  2.5. Cloning of Structural Variant into Expression Plasmid pRSET-A
  2.6. Heterologous Expression using E.coli GJ1158
  2.7. Purification of IL-17E structural variant
  2.8. Biological Activity of rhIL–25
 3. Results and Discussion
  3.1. Amplification of Mutated rh17E
  3.2. Generation of Recombinant DNA
  3.3. Expression of rh17E structural variant
  3.4. Purification of rhIL-17E structural variant
  3.5. Biological Activity Assay
 4. Conclusion
 Acknowledgements
 References

키워드

저자

• Jaya Lakshmi G [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ]
• K Seetha Ram [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ] Corresponding author
• G Ram Mohan [ Department of Microbiology, MVR & PG College, Vishakapatnam-530026 ] Co-first authors
• T Anand [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ] Co-first authors
• P Narindra Kumar [ Department of Microbiology, MVR & PG College, Vishakapatnam-530026 ] Co-first authors
• BK Sreenivas Prasad [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ]
• PVD Soujanya Kumari [ Department of Microbiology, MVR & PG College, Vishakapatnam-530026 ]
• P Balakota Reddy [ Research and Development, Loka Biosciences Pvt Ltd, ALEAP Industrial Estate, Hyderabad-500090 ]
• JB Peravali [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ]
• KRSS Rao [ Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  International Journal of Advanced Science and Technology

• 간기

  월간

• pISSN

  2005-4238

• 수록기간

  2008~2016

• 십진분류

  KDC 505 DDC 605