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DNA cloning and recombinant protein using bacterial expression (questions)

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I have to submit my assignment on time and I need some help. Could someone provide some help or source of reference to me? Thanks.

1.His-tag is well known for its affinity towards nickel and other metal ions. However, other metal-binding proteins present in the cell lysate can also be eluted with our target protein using the present purification protocol (using metal chelating column). How to modify the elution step to improve the purity of the His-tagged fluorescent protein?  

2.Affinity tag may interfere the native function of the target protein. Using recombinant DNA techniques, I can introduce a protease cleavage site into the construct so that the affinity tag can be removable. What are the criteria of selecting the protease to remove the tag? How to design a recombinant DNA construct encoding a removable His-tag fused with a fluorescent protein?

3.The presence of the fluorescent protein was detected by blue light illumination to track the enrichment and purification process of fluorescent protein. What is the advantage of this detection method? And how can we examine the purity of the eluted protein?

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Ninfa, Ballou, and Benore's textbook on the biochemistry and biotechnology laboratory has a good introduction to this topic.

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13 hours ago, BabcockHall said:

Ninfa, Ballou, and Benore's textbook on the biochemistry and biotechnology laboratory has a good introduction to this topic.

Thanks for the information. I will take a look.

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