Protein production is the biotechnological process of generating a specific protein of interest. It is a crucial process that dominates the life sciences, pharmaceutical, and biotherapeutic research.
According to researchers, the main steps in recombinant protein production are:
Derive the cDNA
Create the expression clone and express the protein in an appropriate system.
1.Protein purification and characterization
- Many expression systems are used for protein production, especially those derived from bacteria, baculovirus, mammalian cells, or yeast. However, low protein expression, toxicity, aggregation are few major bottlenecks of protein production. With this article, we will explore elements to improve in the production process to mitigate these inefficiencies.
1.Manage Trace Elements in Cell Culture Media
Trace elements like magnesium in the basal culture could interfere with protein unfolding and folding at the expression phase. Thus, it could lead to contamination/shortfall in the expression of the resulting protein.
To avoid this, researchers should highly optimize the cell culture media. For example, you can produce supplements under cGMP processes with fully marked trace metal levels. Consistency in trace metal levels will reduce or even eliminate variation in the upstream process.
- Detect the Presence of Contaminants With ELISA
The introduction of contaminants is unavoidable during protein production processes. That's where ELISA kits come in. They are highly specific and sensitive, making them the ideal choice to detect the presence of contaminants. Specifically, it is used to investigate markers like host-cell protein (HCP) contaminants and Protein A.
You can improve upstream or downstream bio-processing by using ELISA technology. Moreover, it can help researchers optimize product integrity, maintain cell-line viability, and maximize yield.
3.Improve Protein Solubility by Changing Expression Conditions
When you use strong expression promoters, it can result in high protein concentrations, leading to protein aggregation before folding. You can employ the following condition parameters to enhance protein solubility.
You can lower the inducing agent's concentration to reduce the transcription rate. It will lead to enhanced activity and improvement in protein solubility.
You can increase the protein solubility by lowering the expression temperature (15 - 25°C). At lower temperatures, cell processes like transcription and translation slow down, thereby reducing protein aggregation. In addition, it reduces the degeneration of proteolytically sensitive proteins.
4.Optimize the Codons for Predictable Protein Production
All the amino acids are encoded by multiple codons, implying various tRNAs correspond to one amino acid. Therefore, optimizing codons will increase protein abundance by removing rare codons and replacing them with abundant codons present in the host organism.
5.Minimize the GC Content
A higher GC content at the 5'-end of the coding sequence results in a higher possibility of forming a secondary structure in the mRNA. Therefore, it interrupts translation and results in lower expression levels.
To overcome this issue, you can use A and T residues at the 5'-end in place of G and C without changing the amino acids.
Final Thoughts
The protein production processes are complex, multidisciplinary, and costly. Yet, because of the higher demand for recombinant proteins, the need for innovative and cost-effective bioprocessing strategies is also escalating. Incorporating various methods for improving protein production can have a synergistic effect and enhance transient expression.