You have isolated mRNA corresponding to a protein kinase involved in human cance

Question

You have isolated mRNA corresponding to a protein kinase involved in human cancer. Your ultimate goal is to study the properties of this kinase in an enzymatic assay to understand its function. You will use the tools of recombinant technology and molecular biology to produce a purified sample of this protein. Starting from the mRNA describe a detailed chronological list of all the steps you will follow to obtain a pure protein sample. For each step, describe the rationale/function of the process used.

Explanation / Answer

Answer

Choose the specific expression vector, where the specific DNA segment ( contianing the desried gene) is cloned and that must possess the strong promoter as this synthesize more mRNA which in turn express the desired protein

When the desired DNA segment is cloned and start transcribing into mRNA, it is essential to moniter that it should translate into specific protein product. Therfore, the sample should be seperated from the crude mixture involoving the various tools and techniques. to isolate and purify the protein product

For this purpose, first of all the crude is extracted by ruptureing the cell invoving the use of centrifugation, sonication and filtration. To remove the cell part that surround the target protein.

Now, seperate the fraction on the basis of solubility and on the basis of molecular size and density.

Now, centrifuge the same to obtain the purifed protein

Then, electrophoresis specifcally, SDS PAGE is identify and purify the desired protein

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