Poor Protein Expression in E. coli Despite High mRNA: The Real Reason

When expressing a human gene in E. coli, researchers often find that although mRNA levels are high, protein expression is low or undetectable. This is a classic problem in heterologous expression systems and can occur due to codon usage bias.

The Problem:

You’re observing:

• High mRNA levels (transcription is working)

• Very little or no protein seen on SDS-PAGE (translation is inefficient)

Let’s analyze the given options:

1. Formation of inclusion bodies

   • Inclusion bodies are aggregates of overexpressed proteins, but they would still be visible on SDS-PAGE, just insoluble. This doesn’t explain “extremely poor protein expression.”

2. Lack of human translation initiation factors

   • E. coli uses its own initiation factors. These aren’t required to be human-specific, so this is not a likely issue.

3. Lack of human elongation factors

   • Same as above. E. coli doesn’t rely on human elongation factors.

4. Lack of specific iso-accepting tRNAs

   • Correct. Human genes often use codons that are rare in E. coli. If these codons are not matched by abundant E. coli tRNAs (iso-accepting tRNAs), the ribosome stalls or fails to efficiently translate the mRNA. This is especially true for arginine, leucine, isoleucine codons, etc.

Solution:

To fix this, researchers often:

• Optimize codons in the gene sequence to match E. coli preferences

• Or, use specialized E. coli strains that supply rare tRNAs (e.g., Rosetta strain)

Summary:

Correct Answer:
4. Lack of specific iso-accepting tRNAs

This highlights the importance of codon optimization for heterologous gene expression, especially when using prokaryotic systems to express eukaryotic genes.

Understanding this helps improve protein yields and avoids unnecessary troubleshooting in gene expression experiments.