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Factors affecting protein expression

Update time : 2020-10-27

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Factors affecting protein expression

When doing protein expression experiments, sometimes the experiment feels that there is no problem with the reference experiment method and the cloned protein gene sequence, and the protein electrophoresis has no results. 

The reasons why the protein is not expressed are summarized.

1. Wrong vector construction. This is not uncommon, and many new cloners often mistake the reading frame. For example, Qiagen's pQE series vectors often have a difference of one or two bases in their cloning sites; in addition, some enzymes produce sticky ends and some enzymes produce blunt ends, which can easily lead to reading frame errors and thus fail to express.

2. Improper selection of host bacteria. Different host bacteria have different genotypes. 

For some specially modified vectors, or special purpose vectors, or vectors with special promoters, suitable host bacteria must be selected for expression. 

Therefore, when your protein is not expressed, you can consider replacing the host bacteria.

3. The frequency of codon usage is low. Some genes themselves contain many rare codons, especially the rare codons within 15 bases after the start codon, which have a very important influence on protein expression. Optimized codons seem to have a good effect on prokaryotic expression, but have not seen a good effect on eukaryotic expression systems.

4. The plasmid is unstable or lost. pET systems are usually relatively stable. But when you choose a vector with ampicillin resistance, it may be possible to produce β-galactosidase that degrades the antibiotic and loses the plasmid. 

Another situation is the expression of recombinant toxin protein, which is also toxic to host cells, causing loss of plasmid. This situation is more common in eukaryotic expression systems.

5. The protease degrades the protein. This situation is often caused by the N- or C-terminal sequence of the recombinant protein itself. When the N-terminal of the protein is Arg, Leu, Lys, Phe, Trp, or Tyr, these amino acids are prone to protease degradation, which is the N-terminal rule. 

When the N-terminal is Met, E. coli can steal this Met quietly, especially when Met is followed by an amino acid with a small side chain. 

The presence of non-polar amino acids at the C-terminus can also easily lead to protein degradation. The last 5 amino acids at the C-terminal are polar or charged and are not easily degraded.

6. Secondary translation start site. This is the case when your sequence happens to contain exactly the same sequence as the ribosome binding site. 

The ribosome will find this site and start translating, causing your protein to be truncated, and fragments of the expected size cannot be seen during electrophoresis.

7. SD sequence. The distance between the SD sequence and the start codon sequence (80% is AUG and GUG) also has a very important impact on the efficiency of protein expression. 

The composition of the SD sequence itself also affects translation efficiency. Sometimes in order to reduce the production of inclusion bodies, the SD sequence is specially modified!

8. The secondary structure of mRNA. Before cloning, you have to see if there is a sequence complementary to the ribosome binding site and/or translation start site in your sequence.

9. Unexpected termination. This situation is seen in PCR amplification of sequences. For example, it will mutate the TAC in the middle of the sequence to TAA, which will brake your protein translation. 

Therefore, sequencing must be performed before expression to avoid this situation.

10. Transcription terminator. The existence of transcription terminator can promote protein expression; but when it is lacking, it will cause "read through" and read endlessly. 

This is not a problem in the pET system because it has a selective marker gene in the opposite direction of the target gene. 

If your target gene happens to be in the same direction as this marker gene, then you have to see if there is a transcription terminator after your target gene.

11. The instability of mRNA. The mRNA of the target gene often accumulates in the cell. But the mRNA of coliform bacteria is extremely unstable. 

If a stable structural sequence is inserted in the 5'-untranslated region and 3'-rho-independent terminator of the mRNA, the stability of the mRNA can be promoted. 

Especially if the 5'end has a hairpin structure without protruding, it can make the mRNA inanimate in the cytoplasm.

12. The feasibility of the detection method. Sometimes, the protein is indeed expressed, but the expression level is extremely low, or it is so close to the miscellaneous band that you mistakenly think that the protein is not expressed. 

At this time, you must not forget the two golden rules of biological experiments: control and repetition.

In summary, any experimental materials are very important. The correct construction of protein expression vectors is the basis of protein expression experiments. 

Therefore, the correctness of protein expression vectors must be ensured. A suitable host bacteria is also the basis of protein expression experiments.

If you have problems in the protein expression process, you can ask General Biosystems for help. General Biosystems can provide customized protein expression and purification services to meet most of your protein production needs.