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
1.Bacterial ribosomes were unable to bind to the mRNA corresponding to the Humanprotein of interest
2. Presence of introns in the gene encoding the human protein
3.E. coli RNA polymerase cannot transcribe the sequence encoding the human protein ofinterest
4. Codon bias

 

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Detailed Explanation:

Correct Answer: 4. Codon bias

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What is Codon Bias and How Does it Affect Protein Expression?

When expressing a human protein in a bacterial system like E. coli, one of the most common issues is codon bias. Codon bias refers to the preferential usage of certain codons in a given organism’s genome. This means that different organisms tend to use specific codons more frequently than others to code for the same amino acid. For example, human cells may preferentially use certain codons that are not as common in E. coli.

When a cDNA encoding a human protein is inserted into a bacterial expression vector, the codons in the human gene may not be optimally matched to the bacterial tRNA pool. This mismatch can lead to inefficient translation and thus poor expression of the protein. The bacterial ribosomes may struggle to translate the human mRNA correctly, resulting in low or no expression of the desired protein.

Other Potential Issues:

• Option 1: Bacterial ribosomes were unable to bind to the mRNA corresponding to the human protein of interest:

  • This is unlikely if the vector used has the correct ribosome-binding site (RBS) or if the gene was cloned downstream of a strong bacterial promoter. Most bacterial expression systems are designed to ensure efficient ribosome binding and translation initiation. This issue is more likely related to other factors like codon usage or promoter activity.

• Option 2: Presence of introns in the gene encoding the human protein:

  • Incorrect, because cDNA (complementary DNA) is synthesized from mature mRNA that has already had its introns removed. The cDNA is devoid of introns, making it suitable for bacterial expression systems that cannot process introns. This means that the presence of introns would not typically be the cause of no protein expression in this case.

• Option 3: E. coli RNA polymerase cannot transcribe the sequence encoding the human protein of interest:

  • This is also unlikely. Most bacterial expression vectors are designed with strong promoters (like T7 promoter) that are recognized by E. coli RNA polymerase, ensuring efficient transcription. Unless there is a specific problem with the promoter or plasmid, this is not typically a major reason for lack of protein expression.

Conclusion:

The most likely reason for the failure to express the human protein in E. coli is codon bias, where the human cDNA contains codons that are not efficiently recognized by the bacterial translation machinery. To resolve this issue, codon optimization can be performed to adjust the cDNA sequence to better match the bacterial codon usage, which could enhance translation and protein expression.