Question

How to purify the protein after cloning? Describe each step of the method in detail.

Answer 1

After clonning, it is required to overexpression of gene for target protein. So, I will start with same.

1) Overexpression: Clonned gene is inserted in an expression vector which is then expressed in a host (say E.coli). Expression vector is taken up by host by transformation. Then primary culture is setup for transformed cells followed by secondary culture(1% of primary culture is added as per volume of secondary). This secondary culture is induced using an inducer say IPTG (isopropyl-β-d-thiogalactopyranoside). Then this induced product is harvested.

2)Cell lysis and Protein harvesting: Above induced culture is centrifuged to about 17000*g which then sonicated after pellet is mixed with lysis buffer. Resultant soup is again centrifuged to separate protein (supernatant)and cells debris(pellet).

3) Purification : (There are different techniques for purification I am delivering here Ni-NTA protein purification technique assuming our protein is His Tagged). Coulmn is recharged with resin for Ni-NTA for putification. Then clear lystate from above step is taken into column. Flow through is taken which followed by washing of column 2 times with washing buffer then elution of protein 4 times with elution buffer. For evertime flow through is collected for SDS-PAGE analysis. Protein should be present in flow through of elution step, it shows protein has been purified.

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