Trusted by 10,000+ Scientists Since 2002. View Our 5-Star Google Review, Select Citations and 4,000+ Citations at Google Scholar.

Enhancing Protein Yield In Recombinant Protein Expression Systems

Jan 18th 2024

Enhancing Protein Yield In Recombinant Protein Expression Systems

Recombinant proteins are employed extensively in biological and medicinal research. The expression of recombinant proteins has become increasingly popular due to the development of easy methods available for commercial use. Most importantly, it has dramatically increased the amount of proteins that can be studied structurally and biochemically. Because each protein is unique, purification methods and techniques must be developed for each specific protein while keeping the protein's intended application in mind.

We discuss the numerous parameters that influence protein expression systems and how to modify them to improve their yield in different systems.

What Exactly is a Recombinant Protein?

recombinant protein is described as a customized or modified protein encoded by recombinant DNA

Photo by julien Tromeur on Unsplash

A recombinant protein is described as "a customized or modified protein encoded by recombinant DNA." A plasmid encoding the desired gene in a suitable expression vector is expressed in a particular host expression system to produce a certain amount of the protein for scientific study, therapeutic, or diagnostic purposes.

Considerable applications of recombinant proteins include the following:

  • Study of biochemical processes,
  • Vaccination,
  • A three-dimensional examination of the protein and
  • Applications in biotechnology and therapeutic practices.

Generating recombinant proteins entails replicating the relevant gene into an expression vector while regulated by an inducible promoter.

However, several variables, including proper protein folding, cell growth traits, and appropriate expression indicators at the transcription and translational levels, are necessary to successfully express the recombinant gene. There are numerous possible uses in biotechnology for recombinant protein display on the bacterial surface; however, this requires a deeper understanding of the targeting motifs typically found on carrier proteins that serve as fusion partners.

Also, picking an expression system requires understanding how much it costs to develop, sustain, and other economic factors.

Expression Systems Used in the Production of Recombinant Proteins

Genetic engineering has made it possible to produce valuable recombinant proteins in bacteria

Photo by CDC on Unsplash

Genetic engineering has made it possible to produce valuable recombinant proteins in bacteria, yeasts, insects, mammals, and even plant cells. This biotechnology is already widely used in medicine, and by 2025, its market value is expected to reach USD 400 billion.

Because every kind of host cell is unique, it'snot uncommon for different protein expression methods to produce recombinant proteins with varying biological activity and stability levels. Glycoproteins comprise over fifty percent of human proteins and about a quarter of industrial recombinant pharmaceutical proteins. To extract the most out of these recombinant proteins, eukaryotic cells are usually needed as hosts for glycosylation.

While most commercial biosimilars are manufactured using Chinese hamster ovary (CHO) cells, the expression of therapeutic recombinant proteins is constrained by the vulnerability to feedback inhibition of animal host cells. Furthermore, high manufacturing costs from pricey serum or growth factors and biosafety issues from prevalent animal infections have prompted scientists to pursue nonanimal-derived eukaryotic cell expression methods. Due to their favorable characteristics, including low manufacturing cost, high biosafety, and protein post-modification features, plant cells are preferred for synthesizing pharmaceutical recombinant proteins.

Plant cell expression systems have gained international interest due to the effective expression of numerous biopharmaceutical proteins, such as therapeutic proteins, antigens, and antibodies, currently undergoing clinical studies. Yet, the bacterial expression system remains the leading system at the commercial level.

In our labs at Biomatik, we work with the well-known E. coli system. To generate high yields of recombinant proteins, we must constantly rely on protein expression enhancement techniques.

Bacteria Expression System (BES)

When it comes to producing recombinant proteins, the bacteria Escherichia coli is among the most common hosts. While the bulk of recombinant proteins are created in the cytoplasm of E. coli, disulfide-bonded recombinant proteins are frequently synthesized in the periplasm.

An oxidizing habitat and the resident disulfide bond formation (Dsb)-system can be leveraged to enhance the appropriate disulfide bonding by directing these proteins to the periplasm. Using the periplasm for recombinant proteins with disulfide bonds is better, but the targeting problems of crossing the cytoplasmic barrier can significantly reduce periplasmic yields.

Let's look at the factors and conditions involved in protein expression optimization in bacteria protein systems.

Effects of Protein Sequence and Gene on Solubility and Expression

A prevalent factor contributing to the non-expression of heterologous proteins is the existence of "strange" codons within the mRNA of interest. It is possible to circumvent this codon anomaly through codon-enhanced gene production. One benefit of gene synthesis is that it lets you change the gene's codon bias to work better with the hybrid host.

Expression batches boosted with uncommon tRNAs can surmount the codon anomaly inherent in the recombinant gene for E. coli.

Both expression yield and solubility can be influenced by the residues at the beginning and the end of the target domain. By analyzing the protein's structure and functions, we can find the best places to build the protein domains. By fitting the sequence of the protein of interest onto a homologous protein structure, it is possible to ascertain the suitable domain borders for a protein whose domain structure is unknown. If you don't have access to a homologous protein complex, you should use the forecast of secondary anatomical parts.

Effect of Vector on Solubility and Expression

Proteins from bacteria and other organisms that are bigger usually fold over longer periods

Photo by National Cancer Institute on Unsplash

Certain parts of the DNA sequence, like the Shine-Dalgarno box, promoters, regulatory sequences, transcriptional terminators, sources of replication, and more, control the translation and transcription of the desired gene. Furthermore, selection elements are incorporated into expression vectors to facilitate plasmid selection across the target cell. Including a fusion tag is another essential part of the vector of E. coli expression.

The downstream application and protein target type should be taken into account when choosing a promoter framework. When working with potentially harmful proteins, it's wise to select promoter systems with weak levels of basal output. On the other hand, a more potent promoter must be chosen for the highest possible protein yield improvement. One option to consider for proteins prone to aggregation is to use a cold-shock promoter, allowing expression to occur at cooler temperatures.

Proteins from bacteria and other organisms that are bigger usually fold over longer periods and stick together. In E. coli, folding catalysts and protein chaperones can be employed to prevent proteins from sticking together while making folding easier. Two proteins, one encoded on the target plasmid and the other on an independent plasmid, could be co-expressed.

Fusion tags are attached, at the genetic level, to specific proteins, making them more soluble or easier to find and purify. You usually must try a few different ones to know which fusion tag produces the most soluble proteins. Additionally, the positioning of the tag, which can be positioned at the C-terminus or N-terminus of the protein of interest, is of utmost importance.The majority of fusions involve the N-terminus, and there's an advantage to this type of fusion: it usually improves the expression of soluble protein compared to their C-terminal counterparts.

Considering that the availability of a fusion tag may hinder the biological function of the recombinantly synthesized protein, it could prove useful to enzymatically eliminate the tag following the purification of the fusion protein. To facilitate the elimination of the tag, it is advised to incorporate a division section for a protease unique to a given sequence.

Effect of Host Stocks on Heterologous Protein Expression