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Parameters for expression of protein
I am trying to express human protein in E.coli, but some of them are not getting expressed at all or in a very small amount. I realize that there can be many parameters that affect protein expression at cellular level (like codon usage for example) but I am more interested in parameters in the physiology of cultivation like media and the cell lysis. Today we are using Tryptic soy broth + yeast extract and for cell lysis, lysis buffer. Can a change this two parameters for better protein expression and where can i find resources for this?
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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.
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Dear Ikariotis,

I saw your post just now, I am a molecular biologist and I have tried expression of many mammalian proteins in bacterial system. I found expression will depend upon many factors and not only expression media.
Expression media like TB and LB broth are good for expression.

You may also try-
1- Expression at three different temperatures that is 18, 25 and 37 degree Centigrade.
2- Different time for expression 4 hours, 8 hours and 16 hours.
3- Increase the amount of inducer and try!!!
4- If the protein has any co-factor like Zn, addition of salt related to Zn, like ZnCl2 may help to get better expression.

But I believe that vector system and Host cells are the main issue to get a expression of foreign gene in E. coli system.

BL21 lambda DE3, Rosetta cells etc., may be tried for expression. You must try pET system, pGEX and pQE vector are not good.

When I used some of the mammalian gene, first time I did not get any expression then we have to clone it in many vector systems, some were with tag and some of them without any tag. we have also tried PelB sequence at N and C terminus, we found addition of PelB sequence helps to get better expression and it get auto cleaved, so you may not need to worry about the additional sequence.

I have found hydrophobicity, trans-membrane domain and composition of Amino acid also matters. Proteins with high number of Proline amino acid residues are difficult to express.

Some of the proteins are stable only after formation of di-sulfide bonds and when you express them in bacteria they are not stable. Some of the E coli strains are available in market which makes stable to these proteins.

I would suggest you to get a detail analysis of your protein before selecting a system for expression.

If you have antibody for this protein you may do the western blot and check if it is detectable by western. It will help you to troubleshoot your problem.

Wish you all the best.

Dr. Brijesh N Bhatt
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