How Can Active Protein Expression Be Improved?
The overall role of recombinant proteins in organic and biomedical science is immense; their general importance is identified by the scale of the recombinant protein expansion in the biochemical and structural departments. Since every protein has a different molecular specification, the methods applied for protein purification vary depending on the usage of that particular protein.
Various factors influence protein expression, including the size of the protein. The purpose of the heterologous protein, the organic system from which the protein is extracted, and the expression system of recombinant protein production.
How are Recombinant Proteins Expressed?
The initial knowledge required to initiate the recombinant protein expression successfully is to identify the gene encoding of a specific protein. The application of an appropriate vector is necessary for suitable recombinant protein expression taking into account the cDNA-generated mRNA molecules. Afterwards, an ideal expression system will ensure the smooth expression of the vector.
Some of the preferred protein expression systems are:
- Bacterial Expression System- Escherichia Coli
- Yeast Expression System
- Insect Cell Expression System
- Mammalian Cell Expression System
These methods have their respective advantages and disadvantages. Additionally, their costs and scale of expression also vary. However, the role of these expression structures in antibody production service is immense, with studies highlighting the importance of antibodies derived through various expression systems in research, diagnostics, and therapy.
To understand how the expression process of folded and active proteins can be improved, we must study the effects of the changes.
How to improve Protein Expression?
1. Gene and Protein Sequence Influence
Usually, a “rare” codon in a targeted mRNA plays a detrimental role in developing recombinant proteins. However, you can overcome this challenge with an optimization focused on codon-specific gene synthesis.
Since gene synthesis can transform the codon bias per the specific requirement of the recombinant host, it’s an advantageous option for protein expression. Some studies show that adding rare tRNAs in the protein strains can hugely impact codon bias.
With a higher molecular density of a protein, particularly above the designated weight of above 60 KD, there is an increased chance of failure of the expression. In the E.Coli system, it can be beneficial to the purification process and the overall biochemical activity if the protein is deconstructed rather than using a full-length strain. Additionally, specific fusion tags that enhance the solubility of the protein can be used for this purpose.
2. Vector’s Influence
The usage of various promoters, regulatory sequences, the Sine–Dalgarno box, transcriptional terminations, and replication origins are highly prominent to adequately transcript and translate gene targets of the directed sequential DNA elements.
Additionally, a vector with selected elements helps the plasmid selection in a host cell expression. While choosing an adequate promoter system, the target of the protein and its desired downstream usage enable support during the expression of proteins. The total number of protein yields will depend on the strength of the selected protein promoter.
As mentioned above, fusion tags can play a crucial role in determining the solubility of a protein. Sometimes it becomes necessary to test multiple fusion tags to determine which title will yield the maximum number of soluble proteins.
However, fusion tags interfere with the chemical nature of the recombinant protein; hence, removing the tag after the purification process is crucial.
3. Host Strains’ Influence
Investigators extensively use host strains during protein expression. Many E.Colistrains that match the specific characteristics of the protein are designed specifically for this purpose. Some host strain designs include:
- Proteolysis Susceptible strains
- Strains containing rare codons
- Strains requiring disulfide-bonds
- BL21 strains
Bacterial strains also come with tRNA-encoded plasmids that increase the efficiency of genes with many rare codons. For proteins containing disulfide bonds, expressions using thioredoxin reductase or glutathione reductase will help the solubility by creating cytosolic disulfide bonds.
4. Changing Expression Conditions
A lot of time, the protein expression’s solubility depends on the expression promoter’s conditions. Strong promoter and excessive concentration can lead to protein aggregation before the folding process is completed. A sizable reduction in the transcription rates is necessary for the newly synthesized protein to fold before aggregation. This can be achieved by making alterations in the conditions of the parameter like:
- Dropping the expression temperature
- Lowering the inducer concentration
- Media Choice
5. Improvement in Protein Purification
A well-cushioned solution with ionic strength equal to 300-500 mM of a monovalent salt is used to solubilize and purify the protein properly. Additionally, IMAC (Immobilized metal affinity chromatography) technique is utilized as the first step for protein purification.
However, if your protein requires additional purification, gel filtration and the removal of affinity tags to minimize the non-native sequences can also be used as preferred cleaning methods.
The above study has identified that various internal and external factors affect expression in recombinant proteins. However, it is impossible to achieve optimal protein cultivation without proper investigation.