The growth in the use of recombinant proteins has increased greatly in recent years, as has the wealth of techniques and products used for their amplification and purification. The advantages of using a fusion protein to facilitate purification and detection of the recombinant proteins are now widely recognized. A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant protein needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)6-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor proteinp53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs. Although several tags have been extensively used over the years, his-tags are the unquestionably preferred affinity tag for protein purification.
Name of the Student: Dhivya G
Reg No: 1741210023
Batch: M.Tech II Year
Name of the Guide: Mr. N Manoj Kumar