How to Produce High-Quality Recombinant Proteins

Recombinant proteins have several advantages over their naturally occurring counterparts, including higher yields, more consistent batch-to-batch performance, and an unlimited long-term supply. However, certain features associated with the amino acid sequence may hinder the expression and purification of recombinant proteins. This highlights the importance of having a robust production strategy in place.

Importance of recombinant proteins

Recombinant proteins have broad utility. They may serve as targets for drug development, as cell culture supplements, and as matrices for biomaterial production, and may also be useful for structural biology applications, as industrial catalysts, and as raw materials for diagnostic assays. In addition, recombinant proteins can be used as therapeutics; one of the best-known examples is recombinant insulin, which was first introduced in 1982.

Critical elements for recombinant protein production

Successful recombinant protein production hinges on three key elements:

• The vector—this is typically a double-stranded DNA plasmid containing promoters, multiple cloning sites, and antibiotic resistance genes to both harbor the protein of interest and ensure its transcription
• The expression host—although E. coli is often considered the predominant host for recombinant protein expression, it has limited capability for protein folding and lacks the machinery to add post-translational modifications (PTMs) onto target proteins. As such, advanced eukaryote cells, such as yeast, insect cells, and mammalian cells (HEK293 and CHO) have been developed. Every system has its advantages and limitations, as shown in Table 1.

Table 1. Commonly used host cells for recombinant protein expression.

• The purification system—the purification strategy is dictated by the chemical nature of the target protein. In general, proteins may be purified based on their isoelectric points by passing them through a pH-graded gel or an ion exchange column, or separated based on their size or molecular weight using size exclusion chromatography. Currently, size exclusion-high-performance liquid chromatography (SEC-HPLC) is considered the gold standard for protein purification because of its mild mobile-phase conditions that permit the characterization of proteins with minimal impact on their conformational structure and local environment. SEC-HPLC uses columns containing porous beads to separate molecules based on size, as shown in Figure 1. The advantages of SEC-HPLC over SDS-PAGE for protein characterization are shown in Table 2.

Figure 1. SEC-HPLC for protein purification and characterization

Table 2. Comparison between SDS-PAGE and SEC-HPLC for protein characterization.

It is also important to select an effective method for integrating the target gene. Although E. coli and yeast are amenable to chemical transformation or electroporation, insect cells require an intermediate (usually a baculovirus), while mammalian cells can present unique challenges for protocol optimization. For example, if a mammalian cell line is to be stably transfected, multiple screening strategies must be employed to detect cells with optimal growth characteristics and high yield. And, if the cells are to be transiently transfected, it is essential that the transfection reagent and culture medium are compatible to obtain high transfection efficiencies and protein expression levels.

Strategies for recombinant expression

Using a rational approach for recombinant protein expression is important to obtain a product with the desired purity and yield. Once the protein sequence is obtained, it is recommended to examine basic parameters, such as the molecular weight and isoelectric point, and to identify any key structural features. These may include transmembrane domains, repetitive amino acid domains, or unique PTMs such as lipidations, unusual glycosylation patterns, or a glycophosphatidylinositol (GPI) anchor, any of which could present difficulties during in vitro expression and/or purification.

It is also advised to determine the number of cysteines, because proteins with a high cysteine content may require a more sophisticated expression host to maintain the proper structural disulfide bonds. Other factors to consider are the overall hydrophobicity and general disorderliness of the protein. If certain disordered regions are linked to protein instability, they may need to be removed. In addition, it is important to consider incorporating an affinity tag to simplify protein purification.

After designing the expression construct and identifying a suitable host, pilot studies should be performed along with appropriate controls and functional testing. These small-scale experiments allow time to reconfigure the construct, co-express a chaperone to facilitate protein folding, or select a different expression host to improve the quality and yield of the desired product, as well as provide opportunities for optimizing the purification method. Upon completion of the pilot studies, the process can be scaled up and a suitable formulation identified to maintain product attributes for the intended application.

Challenging targets

Although some proteins are relatively easy to express and purify, others are challenging. These include proteins with one or more transmembrane domains, which usually exhibit low expression and are difficult to dissociate from the cell membrane, and targets that are toxic to the host cell, which limit the amount of biomass available for purification. When working with these types of biomolecules, process design and optimization are especially important.

As a global leader in recombinant technology, Sino Biological is known for manufacturing extremely high-quality and difficult-to-express proteins. With proprietary expression platforms, high-throughput and scale-up capacities, and over 15 years of experience, Sino Biological can successfully develop the target of interest from gene synthesis to protein characterization, no matter how difficult the project. To learn more, please visit sinobiological.com