Proteins are vital molecules in any living organism, dictating different physiological functions and processes. Multiple protein types exist, including structural proteins, catalysts, transportation proteins, enzymes, hormones, and tissue regeneration proteins. Therefore, protein expression is a natural process that gives protein molecules their unique shape and structure, influencing their functionality.
However, proteins have multiple functions outside living organisms, including research and industrial applications. Such applications primarily led to developing recombinant protein expression strategies conducted in vitro using expression systems.
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Protein Expression In The Cell
The cell is the basic unit in all organisms, and proteins facilitate most cellular functions that support life. On the other hand, genes are the primary genetic unit in the body, and each cell contains 23 pairs of chromosomes or long DNA strands containing multiple genes. Consequently, protein synthesis occurs in living cells.
Protein expression begins when environmental factors and cellular signals trigger gene regulation mechanisms to activate specific genes. One literature review defines gene regulation as a process activated to induce or repress the expression of particular genes.
The process controls the type, timing, and volume of genes expressed within particular body cells. Moreover, gene regulation facilitates the production of transcripts or coded genetic material that serves as blueprints during protein expression. Typically, protein expression in the living cell is a two-step process beginning with protein transcription and ending with protein translation. Below is an overview of each process.
As stated above, DNA strands contain genetic material as blueprints for expressing various protein types. Therefore, protein transcription, as the first protein expression stage, begins with a DNA strand. Protein transcription is also a multi-step process and features the following stages.
A DNA strand features a double-helix structure formed by two winding strands, and each strand forming the DNA helix features a gene sequence. Therefore, the double helix’s two strands must unwind to reveal the respective gene sequences.
However, first RNA polymerase, the primary transcription enzyme, binds to a promoter sequence and initiates a transcription bubble to separate the two DNA strands. Promoter sequences are segments of a DNA strand that indicate to RNA polymerase where to begin and end transcription to generate a complete RNA transcript. The promoter sequences typically appear along a DNA strand’s upstream and downstream ends.
Besides RNA polymerase and promoter sequences, transcription factors also have a role to play in protein transcription, forming the transcription complex. Transcription factors are specialized protein molecules that control gene activity, facilitating gene regulation. They bind to the DNA and stimulate the transcription of some sequences while repressing other sequences.
RNA polymerase moves in the 3’ to 5’ direction, creating a complementary RNA (cRNA) from the exposed DNA strand. The enzyme adds complementary or opposite nucleotides to those present on the exposed DNA strand bases. Additional nucleotides elongate the cRNA until it forms a mirror opposite of the exposed DNA strand.
cRNA elongation continues until a protein release factor receives a termination signal from a stop codon. Termination means the cRNA is ready for translation, although additional steps like splicing occur in eukaryotes and capping occurs in eukaryotic cells.
Translation occurs in the ribosomes present in the cell’s cytoplasm. It begins with the RNA polymerase enzyme converting cRNA into a pre-messenger RNA (pre-mRNA) and subsequently into an mRNA.
Second, a transfer RNA (tRNA) molecule carries amino acids to the cell’s ribosomes for translation. The tRNA copies the genetic code present on the mRNA onto the amino acids to form a protein sequence. However, post-translational modifications may occur depending on the target protein’s features.
Protein Expression Systems
Although industrial protein expression occurs in vitro, it requires a live cell to facilitate the expression process. Therefore, protein expression systems are cell cultures that hold a clone of the target gene inside a host cell to facilitate recombinant protein expression.
A protein expression expert puts a plasmid or expression vector with the clone into an expression system. The protein expression systems used in recombinant protein expression include algal, bacterial, mammalian, insect, and yeast.
Each expression system has advantages and disadvantages, depending on the target protein type. Consequently, some expression systems are ideal for specific applications, while others are unsuitable.
As stated above, gene regulation determines the protein types expressed according to environmental signals. Therefore, the expert monitoring expression systems must pay close attention to ensure the system expresses the target protein in the required volumes. The experts use techniques, including the western blot and northern blot, to test for target proteins.
Proteins expressed via expression systems are a complex mix of diverse proteins, cell organelle, debris, and other contaminants. Therefore, protein purification is a necessary protein expression step to help realize the largest biologically active volume of the target protein in its purest form.
Various protein purification methods are available, including affinity chromatography and ion exchange techniques. Such techniques exploit the target protein’s physical and chemical features like solubility and molecular size to separate it from debris and other proteins without denaturing or compromising its integrity. Therefore, purification techniques should match the target protein’s characteristics.
Protein expression is essential for research and industrial applications in multiple industries. Therefore, understanding the process is crucial to choosing recombinant protein expression service providers that meet your project needs.