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Several specialist vendors provide protein expression and production services. Outsourcing this application may save time and effort and bring advantages regarding technical competence and facilities specialized in expressing and synthesizing specific proteins of interest.
Such services can provide researchers with the benefits of a comprehensive workflow that begins with a specific gene and ends with a purified protein and several alternatives for protein expression systems. Among the considerations to consider when selecting a service is the desired expression system, such as bacterial, baculovirus, or cell line.
Proteins regulate all biochemical processes, structure organisms, transport molecules for essential substances, and even act as antibodies to protect the body from outside threats.
Messenger RNA is an intermediary template to transfer the genetic code from DNA to proteins through transcription and Translation. DNA contains the blueprints for proteins, which are subsequently used in Protein Production. Proteins serve an astonishingly wide variety of purposes. Uses for proteins include:
- Create cellular structures, such as the cytoskeleton
- Control protein synthesis to regulate the synthesis of other proteins.
- To make muscles contract, slide along the cytoskeleton.
- Move molecules across the cell membrane
- Enzymatically accelerate chemical reactions
- Serve as toxins
There could be thousands of different proteins in each cell of a living organism, each serving a specific purpose. Their structures differ substantially from their functions. However, they are all linearly ordered polymers of amino acids.
Because proteins comprise 20 different, chemically unique amino acids that form long chains and may be arranged in any order, they have various activities. The structure of the protein affects its ability to perform its intended function.
As the initial step, protein expression seeks to produce numerous proteins. Transcription and Translation are the two primary phases in this gene-to-protein conversion process. It is often accomplished by altering an organism’s gene expression to get it to express a recombinant gene in huge quantities.
Techniques like strain selection, codon optimization, fusion systems, co-expression, mutagenesis, and isotope labeling are frequently employed to obtain sufficient amounts of the desired protein.
Isolating the protein from samples like cell lysates or medium is the next step in the process, known as protein purification. Protein purification involves cleavage of fusion moieties, protein refolding, and chromatography.
Protein chromatographic purification at the research scale is the main topic of the book Protein Purification. A set of procedures designed to separate a few proteins from an intricate combination must typically be used to separate and purify the protein before it can be identified and its features can be examined.
The purification process may first separate the mixture’s protein and non-protein components before separating the desired protein from all other proteins.
Production and Purification of Proteins
Protein output and activity can be maximized by choosing the proper lysis reagents and purification resin. In order to enable the selective purification of the desired protein, most recombinant proteins are expressed as fusion proteins with brief affinity tags, such as polyhistidine or glutathione S-transferase.
A complex system of biotechnologies called “protein production” influences each process phase differently. The features of the recombinant protein and the expression system used largely determine the recombinant protein purification process.
A platform for Producing protein
The biotechnological process of producing a particular protein is known as protein production. Systems for producing proteins from bacteria, insect cells, mammalian cells, and yeast are frequently employed.
The secret to success is choosing the appropriate expression system for your unique application. Characteristics, including protein solubility, functionality, purification speed, and yield, are frequently important when selecting an expression system.
Each system has advantages and disadvantages, which are significant factors to consider when selecting an expression system.
How Protein Production Service Works
Phase 1: Synthesis Of Genes
A codon-optimized sequence generates the target gene in the expression system.
Phase 2: Gene Cloning
An N-terminal signal peptide for protein secretion and an N- or C-terminal 6x Histidine (His) tag for protein affinity purification are added to the target gene when cloned into OVER-expression vectors. DNA sequencing demonstrates the recombinant construct.
Phase 3: Brief Gene Expression
From the cloning cells, plasmid cDNA is collected and transfected into 5 mL of the expression cells. To measure the amounts of gene and protein expression, protein secretion, and protein solubility, scholars analyze the target protein in the cell lysate and supernatant using SDS-PAGE and western blotting with an anti-His tag antibody.
After this stage, the service might be terminated if the protein does not meet your expectations.
Phase 4: Gene Expression and Purification on a Large Scale
Extracted from the cloning cells, the plasmid cDNA is transfected into 2 liters of expression cells. Immobilized metal ion affinity chromatography is used to purify the target protein via its His tag after the cells have been grown. Bidicinchoninic acid assay and SDS-PAGE with Coomassie blue staining are used to measure the concentration and purity of the purified protein, respectively.
The protein is desalted into 1x phosphate-buffered saline, 0.2 m filtered, and pH 7.4. Upon request, the protein can then be lyophilized, aliquoted, and labeled.
Phase 5: Bulk Production
Depending on how much pure protein you need, produce genes on a large scale and purify the resulting protein.
As the quality requirements for protein products rise, the production process becomes more complex, necessitating its development and optimization. With cutting-edge technology and years of experience in protein production, Creative Biostructure now offers technical process development and manufacturing services for expressing and purifying recombinant soluble/membrane proteins.