Earticle

초록

영어

Staphylokinase and its variant (Staphylokinase-Hirulog), the third generation thrombolytic molecule plays an significant role in treating the acute myocardial infarction and stroke which are leading causes of morbidity and mortality across the globe. In refolding of the staphylokinase and its variant from industrially feasible pET28a+, gradual decrease in concentration of urea was used to solubilise the protein. But urea is a chaotropic denaturant, which unravel the structure of proteins by destabilizing internal, non-covalent bonds between atoms. In this study, we selected the expression system to have the increased soluble fraction of sak and its variant from IPTG inducible expression host Escherichia coli BL21(DE3). The expression level and solubility of recombinant sak and its variant from cold shock expression vector pCOLDI was induced at the low temperature (15°C) was highly contrast to the induction process at 37 °C from pET28a+, which gives insoluble fractions. Recombinant sak and its variant were expressed primarily as soluble protein using pCOLDI, later purified with Nickel-chelating resin (Ni-NTA) and the quantity of the purified protein was 913 mg/L. Soluble fractions of purified sak variant having the fibrinolytic activity of 21,825 U/ml and specific anti-thrombin activity of 1,200 ATU/mg. For the first time soluble fractions was achieved instead of inclusion bodies without compromising the quantity and activity of the protein and working with urea was excluded.

목차

Abstract
 1. Introduction
 2. Material and Methods
  2.1. Microbial Cultures and Plasmids
  2.2. Construction of Sak Variant and Cloning
  2.3. Expression and Activity Analysis of Sak Variant
  2.4. Soluble form of the Protein
  2.5. Purification of Recombinant Sak Variant
  2.6. Fibrinolytic Activity of Soluble Sak–Hirulog
  2.7. Antithrombin Activity of Sak–Hirulog
 3. Results and Discussion
  3.1. Generation of Chimeric Staphylokinase and Production of r – DNA
  3.2. Expression and Purification of Sak–Hirulog
  3.3. Fibrinolytic and Antithrombin Activity
 4. Conclusion
 Acknowledgements
 References

키워드

저자

• Seetha Ram Kotra [ Department of Biotechnology, Acharya Nagarjuna University ] Corresponding author
• JB Peravali [ Department of Biotechnology, Bapatla Engineering College ]
• Sudheer Yanamadala [ Hemarus Therapeutics Ltd, Turkapally village, Shameerpet (M) ]
• Anmol Kumar [ Department of Biotechnology, Acharya Nagarjuna University ]
• KRS Samba Siva Rao [ Department of Biotechnology, Acharya Nagarjuna University ]
• KK Pulicherla [ CBST, VIT University ] Co first 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