A cDNA encoding a human protein of interest was cloned in a bacterial expression vector and introduced into bacterial cells for expression. However,…
A cDNA encoding a human protein of interest was cloned in a bacterial expression vector and introduced into bacterial cells for expression. However, no expression of the human protein of interest was obtained. This could be because of
Select an option to attempt
Correct Answer: Option B
Explanation
Codon bias in bacterial expression of eukaryotic cDNA: Different organisms preferentially use different synonymous codons for the same amino acid (codon usage bias). Human cells commonly use codons that are RARE in E. coli (e.g. CGG, CGA for Arg; AUA for Ile; CUA for Leu). When E. coli encounters rare codons during translation: (1) The corresponding tRNA is at low abundance → ribosome stalls; (2) Multiple rare codons cause ribosome dropping off (premature termination); (3) Near-cognate tRNA misreads rare codon → amino acid misincorporation. Result: very low or no protein expression. Solution: (1) Codon-optimise the cDNA for E. coli preferred codons; (2) Use E. coli strains supplemented with extra tRNAs for rare codons (e.g. Rosetta strains expressing rare tRNAs). Note: introns are absent from cDNA (cDNA is made from processed mRNA).