Three expression systems of recombinant protein

Choice of expression vector:

The choice of fusion protein expression vector is restricted by many factors. First of all, there must be a suitable multiple cloning site on the vector. Secondly, it is necessary to select the appropriate fusion tag according to the subsequent protein purification conditions. 

The commonly used fusion tags are His.Tag, GST.Tag, Nus.Tag and Trx.Tag. Among them, the His tag is relatively small and has no obvious impact on the structural properties of the protein. 

After affinity chromatography purification, tag removal is not required; however, a slightly larger tag like GST needs to be removed with a specific enzyme in the subsequent process. In many practical applications, it is often desirable to express a soluble active protein. 

The solubility of a specific target protein depends on many factors, including the vector. The carrier with GST.Tag can increase the solubility of the fusion protein.

Selection of host strain:

The expression status of the same protein in different hosts may be different. The BL21 series hosts are currently the most widely used. The Rosetta host strain derived from BL21 can enhance the expression of eukaryotic proteins with rare codons from E. coli. 

This strain complements the tRNAs of codons AUA, AGG, AGA, CUA, CCC and GGA through a compatible chloramphenicol resistant plasmid. In this way, the Rosetta strain provides a "universal" translation, thereby avoiding expression restrictions due to the frequency of E. coli codon usage. The BL21(DE3) host facilitates the formation of inclusion bodies.

Induction conditions:

Changes in induction conditions also affect protein expression. In general, soluble proteins are mostly needed in practical applications, which requires finding the most suitable induction temperature. 

The proportion of soluble protein synthesized by most proteins under low temperature (15～25℃) overnight induction conditions will be relatively large. 

In addition, the concentration of the inducer also has an effect on the expression state of the protein, and a low concentration of the inducer is beneficial to the expression of soluble protein. Higher temperature may increase the speed of protein synthesis, the speed of folding intermediates to form aggregates, and the hydrophobic interaction force. 

The expressed protein is likely to exist in the form of inclusion bodies. The higher concentration of inducer (0.8-10mM IPTG) can also promote the formation of inclusion bodies in the bacteria. For pET recombinants with ordinary T7 promoters, IPTG with a final concentration of 0.5 mM can be completely induced, while vectors with T7lac promoters require IPTG with a final concentration of 0.8 mM to be completely induced.