Expression and Purification of SAK-fused
Human Interferon Alpha in Escherichia coli |
| Shardul Salunkhe, Bhaskarjyoti Prasad, Ketaki Sabnis-Prasad,
Anjali Apte-Deshpande, Sriram Padmanabhan* |
| Lupin Limited (Biotechnology division), Gat no. 1156, Village
Ghotawade, Taluka Mulshi, District Pune, India 411042 |
| *Corresponding author: |
Dr. Sriram Padmanabhan, Lupin Limited (Biotechnology
division),
Gat no. 1156, Village Ghotawade, Taluka Mulshi,
District
Pune, India 411042,
Tel : +91-20-66549801,
Fax : +91-20-66549807,
E-mail : srirampadmanabhan@lupinpharma.com |
|
| Received December 04, 2009; Accepted December 14, 2009; Published December 15, 2009 |
| Citation: Salunkhe SS, Prasad B, Sabnis-Prasad K, Apte-Deshpande
A, Padmanabhan S (2009) Expression and Purification of SAK-fused
Human Interferon Alpha in Escherichia coli. J Microbial Biochem Technol
1: 000-000. doi:10.4172/1948-5948.1000000 |
| Copyright:© 2009 Salunkhe SS, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. |
| Abstract |
A method for improved refolding and purification of E.
coli derived human Interferon -α (rhIFN α2b) from inclusion
bodies as a Staphylokinase (SAK) fusion protein is
described. Such a fusion protein did not require the supplementation
of rare codons for expression and was found to
be stable at 37°C. The optimal conditions of refolding involved
the use of a mild denaturating agent without the
need for any other agents to prevent aggregation. The SAKrhIFN
α2b fusion protein was successfully purified using
two steps of purification and was cleaved using enterokinase
into two fragments namely SAK and IFN. Both the
proteins were found to be biologically active showing
proper folding of both the fusion partners. The cleaved
IFN showed similar retention time on RP-HPLC as the
bacterial derived untagged purified IFN as well as similar
molecular weight on Agilent 2100 Bioanalyzer indicating
the right processing of the IFN after enterokinase cleavage.
The expression levels of SAK-IFN were found to be
two folds higher than that observed with untagged IFN
under similar experimental conditions. |
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