The Basics of Protein Expression

Protein expression, or gene expression, is central to biological research, molecular biology, and biotechnology. This fundamental process translates genetic information from DNA into functional proteins. Proteins are not only structural components of living organisms, but also key regulators of enzymes, receptors, and biochemical pathways of life. Together with Creative Biostructure, explore the basics of protein expression, discover various expression systems, and learn about our protein expression services.

The Concept of Protein Expression

Protein expression is the process by which genes in DNA are converted into functional proteins. Protein expression involves several steps: transcription, RNA modification, and translation. Each step is intricately regulated by many factors to ensure that the correct proteins are produced.

Figure 1. The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells (The Molecular Biology of the Cell, 6th Edition).

In simpler organisms, such as prokaryotes (e.g., bacteria), this process is relatively efficient. Transcription and translation usually occur simultaneously, thus permitting the rapid production of proteins. However, eukaryotic cells, including plants, animals, and fungi, have a more compartmentalized structure. Here, transcription occurs in the nucleus, while translation occurs in the cytoplasm, with additional RNA processing steps.

The Mechanism: From Gene to Protein

Protein expression follows a well-coordinated sequence of molecular events that ensure the accurate translation of genetic information into functional proteins. The primary stages involved are:

Transcription

The first step in protein expression is transcription, in which the genetic code in DNA is transcribed into messenger RNA (mRNA). This process takes place in the cell nucleus and is catalyzed by the enzyme RNA polymerase. RNA polymerase, also known as RNAP, binds to the promoter region of a gene. The polymerase then unwinds the DNA, using a single strand as a template, and synthesizes a complementary strand of mRNA. The mRNA is then transported from the nucleus to the ribosome in the cytoplasm, where it proceeds to the next step. Transcription is regulated by transcription factors and regulatory proteins that ensure proper initiation and regulation of gene expression.

Figure 2: The process of transcription is carried out by RNA polymerase (RNAP), which uses DNA (black) as a template and produces RNA (blue).

RNA Processing

In eukaryotic cells, mRNA is processed before translation. The introns (non-coding regions) are spliced out and the exons (coding regions) are joined to form the mature mRNA. An additional 5' cap is added to the beginning of the mRNA and a poly-A tail is added to the end. These modifications protect the mRNA, facilitate its export from the nucleus and stabilize it in the cytoplasm.

In prokaryotic cells, RNA processing is minimal compared to the extensive modifications seen in eukaryotic cells. Prokaryotic RNA processing mainly targets ribosomal RNA (rRNA) and transfer RNA (tRNA). Messenger RNA (mRNA), on the other hand, is usually used directly for protein synthesis without significant modifications. In contrast to eukaryotes, mRNA in prokaryotes is usually unprocessed, allowing transcription-translation coupling to occur very rapidly.

Figure 3: RNA processing in prokaryotic cells and in eukaryotic cells. The 5ʹ and 3ʹ ends of a bacterial mRNA are the unmodified ends of the chain synthesized by the RNA polymerase, which initiates and terminates transcription at those points, respectively. The corresponding ends of a eukaryotic mRNA are formed by adding a 5ʹ cap and by cleavage of the pre-mRNA transcript near the 3ʹ end and the addition of a poly-A tail, respectively. (Molecular Biology of the Cell, 6th Edition)

Translation

Once the processed mRNA reaches the cytoplasm, it binds to ribosomes—molecular machines responsible for translating the mRNA sequence into a protein. Translation involves three key stages: initiation, elongation, and termination.

• Initiation: The ribosome assembles at the start codon of the mRNA. Transfer RNA (tRNA) molecules, each carrying an amino acid, bind to the corresponding codon on the mRNA.
• Elongation: The ribosome moves along the mRNA, reading each codon and linking the appropriate amino acids together in a growing polypeptide chain.
• Termination: When the ribosome encounters a stop codon, translation halts, and the completed polypeptide is released.

The resulting polypeptide chain then undergoes further folding and modifications to become a functional protein, ready to perform its biological role.

Figure 4: Initiation and elongation stages of translation involving RNA nucleobases, the ribosome, transfer RNA, and amino acids.

The Biological Importance of Protein Expression

Protein expression is critical for the function of cells and organisms. Proteins are involved in almost every cell process: as enzymes to catalyze metabolic processes, as structural molecules to shape and support cells, and as signaling molecules to help cells communicate between cells and their environment. Proteins are also involved in cell homeostasis and stress. For example, when cells are under environmental stress, they may increase the production of specific proteins to help them survive and recover. Conversely, over- or under-expression of proteins is associated with disease.

In cancer, for example, mutations can cause the overexpression of proteins that promote uncontrolled cell growth. In contrast, in neurodegenerative diseases such as Alzheimer's or Parkinson's, proteins are misfolded or aggregated, disrupting cellular function and causing tissue damage. The regulation of protein expression is, therefore, crucial for maintaining cellular balance and overall organismal health.

Protein Expression Systems in Biotechnology

Given its fundamental importance, understanding and manipulating protein expression is crucial of many biotechnological and industrial applications. Protein expression systems are designed to mimic or enhance the natural processes of protein synthesis, often using engineered cells to produce large quantities of proteins efficiently. There are several key types of protein expression systems, each with its strengths and limitations.

Prokaryotic Systems: E. coli

Bacterial systems, particularly Escherichia coli, have long been a popular choice for protein expression. They are relatively inexpensive, easy to cultivate, and capable of producing large quantities of recombinant proteins quickly. These systems are particularly useful for producing simpler proteins that do not require extensive post-translational modifications.

However, one major limitation of bacterial systems is their inability to perform complex modifications, such as glycosylation, which is essential for some eukaryotic proteins. Furthermore, bacterial systems are not suitable for proteins that require correct folding or those that are toxic to the bacteria.

Eukaryotic Systems: Yeast and Mammalian Cells

Eukaryotic systems, such as yeast and mammalian cells, are used to produce proteins that require more complex post-translational modifications. Yeast, a single-celled eukaryote, provides a balance between the simplicity of bacteria and the complexity of mammalian cells. Yeast cells can perform some glycosylation and other modifications, so they're an ideal option for producing some types of therapeutic proteins.

Mammalian cell systems, such as Chinese hamster ovary (CHO) cells, can produce complex proteins, particularly therapeutic monoclonal antibodies and other glycosylated proteins. These systems are more expensive and slower than bacterial or yeast systems. They have the advantage of producing proteins that are structurally and functionally similar to those produced by humans.

Cell-Free Systems

In addition to cellular systems, cell-free protein expression offers a novel approach to producing proteins without the use of living cells. Cell-free systems use cell extracts (from sources such as E. coli or rabbit reticulocytes) to perform transcription and translation in a test tube. These systems are highly flexible and efficient, allowing rapid production of proteins without the constraints of cellular growth cycles. Cell-free systems are particularly useful for high-throughput screening, the production of small amounts of protein for structural studies, or the synthesis of toxic proteins.

Figure 5: Various protein expression systems.

Challenges in Protein Expression

While the potential of protein expression systems is vast, several challenges can arise in their application. These include:

• Codon Bias: Different organisms tend to prefer certain codons over others. When a gene is transferred from one organism to another, these differences in codon preferences can make it harder for the new host to produce the protein efficiently.
• Protein Misfolding: Some proteins are prone to misfolding or aggregation, especially when produced in large quantities. This can result in the production of proteins that are either inactive or toxic, making purification and application more difficult.
• Post-Translational Modifications: Many proteins require specific modifications for their function, such as phosphorylation or glycosylation. However, bacterial systems often have difficulty carrying out these modifications, which limits the range of proteins they can produce.

Applications of Protein Expression

The applications of protein expression are vast and varied. In the pharmaceutical industry, recombinant proteins produced by engineered expression systems are critical to the production of vaccines, monoclonal antibodies and other biologics. In research, protein expression systems allow scientists to study the structure and function of proteins, paving the way for drug discovery, enzyme engineering, and more.

In summary, protein expression is the foundation of biological systems and holds immense potential for scientific and industrial advancement. Creative Biostructure provides cutting-edge protein expression services to a wide range of industries, driving innovation in biotechnology and research. Contact us today to explore customized protein production solutions to meet your specific needs!

Reference

1. Molecular biology of the cell (6th edition, 2015). Garland Science, Taylor and Francis group.