Tips On Optimizing Protein Production

In recent years, with a significant amount of advancements, the system of recombinant proteins have gained visibility on the commercial front. Researchers and scientists use recombinant proteins in biomedical and biological science. Furthermore, the commercial system’s development has affected the production system of recombinant proteins. Such a process has allowed the more number of proteins to be investigated structurally and biochemically. It is essential to understand that every protein has a considerable role and difference. As a result, purification strategies and protocols should come under the system to study every protein, keeping its use in mind. 

Since protein production and its optimization are unique, here is a rundown of some of the tips that you can incorporate into optimizing protein production systems. 

1. Vector’s influence on solubility and expression

Amid the DNA sequence process, several elements work together to directly translate and transcribe target genes. These elements are regulatory sequences, promoters, transcriptional terminators, the Shine-Dalgarno box, and the replication’s origins, etc. Moreover, vectors of expression comprise a vast selection element. This takes place to accommodate plasmid selection amid host cells. Fusion tags are another vital feature of the E. coli expression.

While optimizing the process of protein production, you need to select a promoter system. However, your protein target’s nature and downstream should also be finalized and considered to move further. If you find your protein target to be toxic, use a promoter system with low basal expression. However, if you are operating on maximal protein yields, select a robust promoter. And, for proteins that are aggression-prone, go for the cold-shock promoters.

2. Understand the native conformation

One of the essential things that you need to know about the protein production system is its native conformation. If you tend to perform one of the feasibility studies alongside bio-informatics, you can ease its designing process. When you understand the native conformation, you will be able to determine disulfide bridges, obtain solubility, identify glycosylation sites, analyze predictive secondary, stability probabilities, and then tertiary structures.

In such a way, projects that begin with an in-depth analysis always have the potential to succeed. Furthermore, it allows you to select a processed production system, such as:

• Utilization of bacterial strains that are capable of forming several disulfide bridges amid their unstable proteins or cytoplasm toxic
• Utilization of human cells (HEK293 EBNA cells) to manufacture functional recombinant proteins that are the closest to in-vivo context. You can perform such a process because of the post-translational modifications and the availability of chaperone proteins.

3. The impact of host strains on heterologous protein’s expression

The development of bacterial host strains helps in supporting heterologous proteins’ expressions. E. coli strains that are commercially available undergo a specific designing process for unique protein expressions. Furthermore, they are vulnerable to proteolysis and contain several rare codons, or need disulfide-bonds.

• Experts recommend that you use protease deficient strains like E. coli BL21 or its other derivatives for recombinant proteins vulnerable to proteolytic degradation.
• All the distinctions found in codon frequency between the expression host and the target gene can result in premature translation termination, translational stalling, and the misincorporation of amino acids. You can overcome such a difference by the procedure of supplying rare tRNAs during the system of expression. You should also promote bacterial strains comprising plasmids that have the potential of encoding rare tRNAs. This can be done through the process of efficient expression of genes with high rare codons’ frequencies.

4. Change expression conditions to improve proteins’ solubility

The utilization of high inducer concentrations and strong expression promoters might lead to concentrations of high protein. This can further result in protein aggregation before the method folding comes into the picture. If you happen to reduce the costs of translation or transcription, it will help facilitate the folding system by allowing synthesized proteins to come under folding before aggregating. One of the standard parameters that you can manipulate to optimize protein solubility is temperature.

In terms of temperature, lower expression temperatures (15-25°C) can optimize recombinantly expressed protein solubility. When it comes to lower temperatures, cell processes tend to slow down, leading to minimized rates of translation, transcription, reduced protein aggregation, and cell division. Furthermore, low expression temperatures can also lead to reducing proteolytically sensitive proteins’ degradation.

Additional steps to improving protein purification

Purify and Solubilize proteins in a well-buffered mixture. The solution/mixture should contain an ionic strength equal to 300–500 mM of monovalent salt, like NaCl. You can utilize immobilized metal affinity chromatography (IMAC). Make sure that it is your first purification step. In case you require additional purification, try using size-exclusion chromatography. Furthermore, as a concluding step, you can utilize ion-exchange chromatography. You can remove the affinity tag to minimize sequences non-native in nature amid a recombinant protein. This can also result in achieving further purification.