Escherichia coli has become a popular organism of choice in recombinant protein production services. It is mainly used as a cell factory because it is very established, thus becoming a popular expression platform. Because of that, there are many molecular protocols and tools available at hand for the heterologous’ high-level production. An example is the vast catalog of expression plasmids which comprises many cultivation strategies and many engineered strains. Let us look at the various approaches for synthesizing recombinant proteins in E.coli and the latest progress.
Today, the popular expression plasmids used in protein production services result from various promoters, replicons, selection markers, multiple cloning sites, and fusion protein or fusion protein removal services. Because of that, there is a massive catalog of existing expression vectors, making it an uphill task to choose the most suitable one.
Genetic elements undergoing replication as autonomous units like plasmids possess a replicon. A replicon consists of one replication origin alongside its associated cis-acting aspects of control. When choosing a suitable vector, one of the most critical parameters to consider when selecting an appropriate vector is the copy number. The copy number control resides in the replicon. Logically, the high plasmid dosage equates to a more yield of recombinant proteins as many units of expression make residence in the cell.
However, a high number of plasmids will impose a metabolic burden that decreases the rate of growth of bacteria producing plasmid instability, making the number of healthy organisms for the synthesis of proteins fall—because of that, using a high copy number of plasmids for the expression of proteins implies the production yield.
The lac promoter is the staple for prokaryotic promoter research, which is also the main component of the lac operon. There has been an accumulated knowledge of the system’s functioning, which has led to its extended usage in the expression vectors. Lactose causes an induction from which the sugar could be used for protein production. However, in readily metabolizable carbon sources, the induction process is complicated. In the presence of glucose and lactose, expression from lac promoter is not entirely induced until the utilization of all glucose.
At that point, the production of cyclic adenosine monophosphate takes place-it is necessary for the lac operon’s complete activation. That positive expression control is called catabolite repression.
A resistance marker is added to a plasmid backbone to deter plasmid-free cells’ growth. In the systems of E.coli, the genes of antibiotic resistance are used habitually for that purpose. The bla gene confers resistance to ampicillin, and its product is a periplasmic enzyme inactivating the β-lactam antibiotic’s β-lactam ring. However, the β-lactamase undergoes a continuous secretion, and the antibiotic degradation ensues for many hours until ampicillin is almost finished or depleted.
The cells that do not carry the plasmid under this situation are allowed to increase their number in cultivation. The selective agents whose resistance is based on degradation, such as kanamycin and chloramphenicol, have this problem. Thus, tetracycline is very stable during cultivation since resistance is based on the active efflux of resistant cells’ antibiotics.
When you devise a project where there is a need for a purified soluble active recombinant protein, it is indispensable to possess the means for the following:
- Detecting it along with the purification and expression schemes
- Attaining the maximal solubility
- Efficiently purifying it from the cellular milieu’s E.coli
A stretch of amino acids or the peptide tag’s expression along the desired protein forming a chimeric protein allows the three goals above to be attained straightforwardly. Peptide tags are small and thus less likely to interfere if they are fused to the protein. In some cases, though, they could negatively affect the biological activity or the tertiary structure of the fused chimeric protein. The vectors that allow the tag positioning on either the C-terminal or the N-terminal end are available. They are positioned at the N-terminal end for secreting recombinant proteins.
If there is a need for biochemical or structural studies on recombinant proteins, the fusion partner should be eliminated from the recombinant protein. Additionally, peptide tags need to be removed since they could interfere with protein structure and activity. They could sometimes be left in place for the sake of crystallographic studies. Tags are typically eliminated using either chemical cleavage or enzymatic cleavage.
Obtaining a good amount of soluble protein is usually not the road end. That is because the obtained protein may still be of bad quality, among other inconsistencies. One of these inconsistencies includes the incomplete folding, which is always the culprit in this case. In such a case, the protein usually adopts a stable soluble conformation. However, the exact architecture of the site is still entirely unsuitable. In this case, some options which have been addressed can be helpful. Some proteins need small prosthetic groups or molecules to acquire their final folded conformation.
Adding these compounds to the culture media increases the quality and the yield of the expressed protein significantly. Additionally, the production of proteins at lower temperatures profoundly impacts the quality of the protein. The conformational functionality and quality of the recombinant proteins that are highly soluble increase with the reduction of the temperature in the culture.