Question

Imagine you want to study one of the human crystallins, proteins present in the lens of the eye. To obtain sufficient amount of the protein of interest, you decide to clone the gene that codes for it. Assume you know the sequence of this gene. How would you go about this? In a paragraph of approximately 3 to 5 complete logical sentences, explain the technique that you would use, why, and its basic steps.

To answer this question you need to state whether the following statement is true or false and state two reasons why you think so:

A eukaryotic gene, beginning at its transcription start site and ending at its transcription termination site, that is 90,000 bases long will lead to a protein that is 30,000 amino acids long.

Answer 1

The statement is false. As the protein formation only start from the start codon (AUG) and ends at the STOP codon (UAA, UAG, UGA), and do not depend on the transcription start/ termination site, the protein formed can never be same size as the mRNA sequence size. It will always be shorter than mRNA size.

The human genome has a lot of non-coding sequences present in the genes required for gene regulation. The eukaryotic genes contain exons and introns, which upon splicing of mRNA results in the cutting of introns and joining of exons, form a protein. eg if a gene is 90,000 bp long, only 1% of that sequence may be exon and rest is introns.

As mentioned earlier, the mature mRNA is formed after many post-transcriptional modifications like splicing. So for cloning of human crystallin proteins,

1. Take the sequence of mRNA and design primers with appropriate restriction enzyme sites compatible with the chosen cloning-expression vector.
2. Isolate mRNA from human cells and perform reverse transcription to convert it into cDNA, as we can only use DNA for PCR reaction.
3. Use the cDNA from the second step to perform PCR with the designed primers and obtain the amplified DNA product of human crystallin (insert for cloning).
4. Perform restriction digestion of both insert and chosen vector and ligate them together.
5. Transform the ligated product into E. coli to select clones.
6. Once clone is selected, transform into E. coli expression cells and purify the expressed protein.