Intein Mediated Protein Synthesis and its Purification by Ni- NTA Affinity Chromatography

 
 
International Journal of Biotech Trends and Technology (IJBTT)
 
© 2015 by IJBTT Journal
Volume - 5 Issue - 2                         
Year of Publication : 2015
Authors : U.Preethi, Dr. Tha.Thayumanavan

U.Preethi, Dr. Tha.Thayumanavan "Intein Mediated Protein Synthesis and its Purification by Ni- NTA Affinity Chromatography", International Journal of Biotech Trends and Technology (IJBTT), V5(2):38-42 Apr - Jun 2015, Published by Seventh Sense Research Group.

Abstract

An intein mediated protein purification system was established to generate His-tag free protein with high level of purity. To achieve this a synthetic intein gene (~171 bp) was constructed and fused to a histidine sequence at the C-terminus by a two-step assembly PCR. The synthetic intein gene was inserted into pEt-32a(+) vector at ECoRV and HindIII region. The target gene MBP from pMal-c5E vector fused in frame at the Hind-III and Xho I site of pEt- 32a(+), containing the intein gene. The plasmid was transformed into E. coli BL21 (DE3) strain and protein expression was induced by IPTG. The MBP-Intein-His tagged fusion protein was purified from cell lysates using Ni-Nta resin affinity chromatography. The MBP was removed from intein - fused pEt vector by on-column intein-mediated cleavage with DTT.

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Keywords
intein, histidine tag, MBP, pMal-c5E, pET32a (+), E. coli BL21 (DE3), Ni-Nta.