08-24-2013, 04:03 AM
Protein expression is a biotechnological technique that is used by researchers to study the translation mechanism and protein presence and abundance in a cell. It has been used to study mainly for drug discovery purposes.
Protein expression in Escherichia coli:
Protein expression systems can be of many types out of which cell based expression systems are the oldest and most common. E.coli is one of the most favoured host systems. Transcription of a gene and translation of the resulting mRNA require specific signals both upstream and downstream of the coding sequence of the gene. In case of bacteria, the upstream sequences required are promoter (P), and ribosome binding site ® while the downstream sequence is transcription terminator (T). An expression vector contains the P,R, and T sequences in a cluster, which commonly are termed as expression cassette.
Factors affecting protein expression:
- Number of copies per cell of the expression vector
- overall stability of the expression vector ( under selection of the vector); smaller vectors are more stable than the larger ones
- Promoter strength; stronger the promoter, higher is the expression.
- stability of the heterologous mRNA; this is one of the major rate determining factors. It should be noted that mRNA half-life cannot be accurately predicted from its base sequence; therefore, has to be empirically determined.
- The presence of correct transcription termination signal at the 3’- end od mRNA; it enhances mRNA stability.
- The 5’- region of mRNA should be optimized for translation in yeast. For example, the presence of an upstream AUG in the mRNA leader sequence effectively inhibits translation initiation in yeast, while it is immaterial in mammals.
- Recombinant protein degradation in the cells and during downstream processing; the problem can be minimized by using protease deficient strains as host.
- Extracellular secretion protects recombinant proteins from proteolysis. Secretion can be achieved by adding the appropriate short hydrophobic signal sequence at the N-terminal end of the recombinant protein.
-affinity tag used,
-size and source of the heterologous protein,
-codon biasing,
-E. coli strain used,
-culture conditions and
-protein degradation
In answer to your question, though Luria Bertani broth is the most used medium in many academic labs and it gives high growth and propagation of the bacteria, it doesn’t give high protein production. Terrific broth or Super broth are more suited for higher rate of protein production. Another way to increase production with LB broth is addition of Magnesium to the medium and increased agitation.
Protein expression in Escherichia coli:
Protein expression systems can be of many types out of which cell based expression systems are the oldest and most common. E.coli is one of the most favoured host systems. Transcription of a gene and translation of the resulting mRNA require specific signals both upstream and downstream of the coding sequence of the gene. In case of bacteria, the upstream sequences required are promoter (P), and ribosome binding site ® while the downstream sequence is transcription terminator (T). An expression vector contains the P,R, and T sequences in a cluster, which commonly are termed as expression cassette.
Factors affecting protein expression:
- Number of copies per cell of the expression vector
- overall stability of the expression vector ( under selection of the vector); smaller vectors are more stable than the larger ones
- Promoter strength; stronger the promoter, higher is the expression.
- stability of the heterologous mRNA; this is one of the major rate determining factors. It should be noted that mRNA half-life cannot be accurately predicted from its base sequence; therefore, has to be empirically determined.
- The presence of correct transcription termination signal at the 3’- end od mRNA; it enhances mRNA stability.
- The 5’- region of mRNA should be optimized for translation in yeast. For example, the presence of an upstream AUG in the mRNA leader sequence effectively inhibits translation initiation in yeast, while it is immaterial in mammals.
- Recombinant protein degradation in the cells and during downstream processing; the problem can be minimized by using protease deficient strains as host.
- Extracellular secretion protects recombinant proteins from proteolysis. Secretion can be achieved by adding the appropriate short hydrophobic signal sequence at the N-terminal end of the recombinant protein.
-affinity tag used,
-size and source of the heterologous protein,
-codon biasing,
-E. coli strain used,
-culture conditions and
-protein degradation
In answer to your question, though Luria Bertani broth is the most used medium in many academic labs and it gives high growth and propagation of the bacteria, it doesn’t give high protein production. Terrific broth or Super broth are more suited for higher rate of protein production. Another way to increase production with LB broth is addition of Magnesium to the medium and increased agitation.
![[+]](https://www.biotechnologyforums.com/images/netpen-pro/collapse_collapsed.png)