You want to purify large amounts of a human tumor suppressor protein called Tusu

Question

You want to purify large amounts of a human tumor suppressor protein called Tusup for biochemical studies. The easiest and cheapest way to achieve this is to place the Tusup gene in the bacteria E. coli. Unfortunately, you find that the bacteria produce only a small amount of Tusup. Even worse, they produce far more N-terminal fragments than full-length Tusup proteins. Your advisor notes that your protein has many arginine codons and this may cause a problem. You peruse the literature on codon usage in bacterial and human genes and discover that human genes have a bias for the various arginine codons that differs from that of E. coli genes, as shown in the Table. Why might this different codon usage limit the production of full-length Tusup protein? How might you remedy the problem? (Note: this is a problem experienced by molecular biologists and chem engineers trying to express human proteins in E. coli)

Explanation / Answer

Why might this different codon usage limit the production of full-length Tusup protein?

Fast growing microorganisms like E.coli or S. Cerevisiae will have preference for some codons and this reflect the composition of their genomic tRNA pool. This will help them to go for faster translation rates with high accuracy. Humans do not show high growth rate, so they don’t have codon usage optimization. Within a genome, the occurrence of synonymous codons are not equal and some synonymous codons occur much more frequently than others. It also has observed that preferred codons differ among different species. This is mainly based on the GC content of the organism, relative amounts of isoaccepting tRNA molecule, amino acid conservation and RNA stability.

So, the DNA sequence what we are using to encode polypeptide will have great impact on the way it will be translated inside the bacterial cell. So when you insert transgene, the bacteria may translate it with less efficiency because of the presence of rare codons along the transcript. But during this time it will produce abundant amount of its own protein, thus causing the depletion of ribosomes. So, in some cases the protein will not be completely synthesized.

How might you remedy the problem?

We can go for the codons which are already the organism has optimized for tRNA pools, this can increase the expression of transgene. We can also go for codon usage to get more of our protein from the bacteria.

There are two strategies for solving codon usage bias

(a)Codon optimization of the foreign coding sequence

(b)Increasing the availability of underrepresented tRNAs by host modification

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