The production of recombinant proteins is one of the most powerful methods in life sciences. Its creation and purification can lead to many industrial processes and the diagnosis and treatment of diseases. Various advances and challenges come with recombinant protein expression in Escherichia coli (E. coli). At first, expressing recombinant proteins may seem straightforward.
Cloning of the DNA that encodes a target protein’s code occurs downstream of the promoter. Elisa Kit Manufacturers allow for the easy, convenient, and reliable study of unstable protein expression, dissolved protein biomarkers in several matrices. These matrices include serum, plasma, and cell culture supernatants or lysates. Introducing this vector into a host cell, and the cell’s protein-synthesis machinery produces the desired protein.
There are tips to increase unstable protein expression. This write-up discusses them. Read on to find out.
1. Increasing unstable protein expression influence of host strains
To support heterologous protein expression, we have to create bacterial host strains. We engineer the commercially available strains of E. coli to express proteins that are proteolytic-sensitive. These strains require disulfide bonding and contain rare codons. We recommend using protease-deficient strains, such as E. coli BL21 and its derivatives for proteins susceptible to proteolytic degrading.
Differences in the codon frequency between the target and expression hosts can cause translational stalling and premature translation termination. We can subdue the difference by providing rare tRNAs for expression. Bacterial strains containing plasmids encoding rare tRNAs will promote the efficient expression of genes with a high frequency of rare codons.
The expression will assist proteins with disulfide bond formation in the thioredoxin reductase strains (trxB) or glutathione reductase host strains. They will increase the solubility of the folded disulfide-containing protein. Another strategy for disulfide-containing protein expression is to target the expressed protein at the E. coli periplasm. This method is highly oxidative and promotes disulfide bond creation.
2. Increasing unstable protein expression by changing the sequence
Sometimes, intra-RNA interactions can prevent the optimal translation of recombinant gene expression studies. The secondary structure of the untranslated areas, the ribosomal bound site, and the affinity tag code sequence can cause problems. The presence of particular codons can cause expression problems within the gene of concern.
It is possible to increase the expression of genes with complex secondary structures by changing ribosomal binding sites and removing secondary inhibitory systems. For improved expression, it is common to convert ribosomal sites to be more similar to the E. coli sequence AGGAGG. Sometimes, we can enhance expressions by changing the position of an affinity tag and adding more adenines at the next codon following the initiation codon.
3. Increasing unstable protein expression by a tunable expression of toxic proteins
Researchers often require a tunable expression for toxic proteins, which needs strict promoter control. By keeping the target protein’s concentration below the tolerance of the host strain, you can maximize yields and keep expression at the desired level.
Tuning expression can also stop well-expressed target proteins from creating inclusion bodies.
PrhaBAD promoters are a critical part of many expression systems. It may be necessary to use a cell-free expression method, such as the Purexpress In Vitro Protein Synthesis Kit, to express highly toxic proteins.
4. Increasing unstable protein expression by changing expression conditions
Strong expression promoters and high inducer concentrations can cause increased protein concentrations, which may cause protein aggregation before folding. The reduction of transcription and translation rates will make folding easier. Many of these conditions can lead to increased protein solubility.
A lower expression temperature (15-25 DegC) will improve the solubility of recombinantly derived proteins. Lower temperatures cause cell processes to slow down, leading to decreased transcription, translation, and protein aggregation rates. We can also achieve a reduction in proteolytically sensitive protein degradation by lowering the expression temperature.
● The concentration of the inducer
We can reduce the transcription rate if the induction agent is lower. The low induction rate will also improve the solubility of recombinant protein and their activity.
● Media choice
Batch culture is the most popular method for cultivating cells for recombinant protein expression. We must include all nutrients in the growth medium from the beginning.
5. Increasing unstable protein expression through the Influence of vector
Promotors, regulatory sequences, and the Shine-Dalgarno Box are all DNA sequence elements that control the transcription and translation target genes. Expression vectors also contain a selection element that aids in plasmid selection within a host cell. The presence of fusion tags is another critical feature in the E. coli expression vector.
We must consider the nature and downstream uses of the protein target when choosing a promoter system. Consider using promoters with low basal expression if the protein target is toxic. To increase the solubility of proteins, we genetically fuse fusion tags to target proteins. We may test multiple fusion tags to determine which one yields the highest amount of soluble protein.
It is essential to place the tag at either the N-terminus (or C-terminus) of the target protein. The most popular N-terminal and C-terminal Fusions are more effective than C-terminal.
Some proteins are challenging to express in a heterologous host. Recent developments in tunable solubility technology and protein targeting have made it possible to improve unstable protein expression. These systems are significantly more efficient than those used a few years ago.