Protein production is a naturally occurring process in all living cells since they are essential for many life-sustaining biological processes. It occurs in a two-step process that involves the transcription of a code from DNA onto an mRNA, followed by the translation of the mRNA into protein molecules.
However, advances in biotechnology facilitate custom protein production through processes like recombinant technology, where researchers select specific genes as protein templates to create novel proteins. Recombinant protein production supports research for medical advancements through the production of medication, vaccine antibodies, and artificial enzymes like insulin. Below is a step-by-step breakdown of how recombinant protein production occurs.
1. Identify The Gene of Interest and Create a cDNA
Natural protein synthesis entails the formation of a messenger RNA (mRNA) from a DNA strand. It begins when the enzyme RNA polymerase causes the DNA helix to unwind partially, revealing the gene sequence to be copied onto the RNA.
However, recombinant protein production begins with a complementary -DNA (cDNA), a structure opposite to the DNA. cDNA formation occurs when an enzyme called reverse transcriptase (RT) copies the gene sequence from an RNA onto a cDNA and converts the single-stranded RNA into a double helix like the DNA.
The cDNA and annotated DNA facilitate artificial manipulation of the RNA during biological research on gene sequencing. DNA annotation refers to identifying genes along various coding regions of the DNA and establishing their specific functions.
By DNA manipulation, scientists created, updated, and maintained a repository of 19,000 cDNAs containing annotated genes, ideal for recombinant protein production. Furthermore, scientists can also use gene synthesis to create cRNAs that best suit specific gene expression methods.
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Now that you have a suitable cRNA for recombinant protein production, the next step is to clone it. Cloning in biochemistry refers to making multiple copies of a specific portion of the DNA, or cRNA.
During recombinant cloning, a target gene or gene of interest gets “cut” from the cRNA and pasted inside a cloning vector called a plasmid. Note that “cut and paste” enzymes, namely restriction enzymes and DNA ligase, direct the movement of the target gene fragment from the cRNA to the plasmid.
Restriction enzymes recognize the target area on a cRNA and cleave or cut the specific portion off. They precede the enzyme ligase that functions by repairing single-stranded breaks and forming chemical bonds between the target genes cut by the restriction enzymes. Therefore, the two enzymes generate a recombinant plasmid ready for recombinant protein expression.
3. Recombinant Protein Expression
The recombinant plasmid must go through an expression system to form recombinant proteins. Expression systems are hosts for the recombinant plasmid and culture them to synthesize the desired/target protein. The four central protein expression systems that researchers use during recombinant protein expression are:
- Bacterial expression systems
- Yeast expression systems
- Insect expression systems
- Mammalian systems
Each recombinant protein expression system has unique pros and cons regarding how they replicate proteins. The general considerations when selecting the ideal expression system include:
- Compatibility between the recombinant plasmid and the host
- Expression system ease of use
- Duplication speed
- Cost of requisite machinery and scaling up
- Enhanced efficiency of recombinant proteins
Bacteria and yeast are the ideal expression systems for the mass production of proteins at a minimal cost; they are also the most user-friendly expression systems. However, the mammalian system is best for proteins that require additional processing after synthesis. Therefore, consider the protein function and intended use before selecting a protein selection method.
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4. Recombinant Protein Conformation and Testing
Recombinant protein conformation involves conducting a test to establish the target protein presence after the recombinant plasmid spends the appropriate time in an expression system. The most common test performed is a PCR (polymerase chain reaction) test that involves detecting genetic material in the culture medium.
Another recombinant protein confirmation test is the SDS-PAGE method that functions through the separation of molecules. Different researchers use different methods to confirm recombinant protein presence.
5. Protein Purification and Isolation
After the protein confirmation test establishes recombinant proteins’ presence, the next step is to separate protein molecules from non-protein molecules. Later, protein isolation occurs whereby the researchers separate target protein molecules from other synthesized recombinant protein molecules.
Similar to the SDS-PAGE method, protein isolation also relies on distinctive molecule characteristics to separate protein molecules from the culture medium. The main techniques employed in protein isolation include solvent use, ion exchange, tags, and centrifugation; protein solubility and their location in the medium culture dictates the appropriate method. Second, the recombinant proteins also require purging of toxins like pyrogens present in the host medium. Effective protein purification occurs under a specialized machine called a spectrophotometer.
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6. Protein Characterization
Protein characterization involves a series of tests to determine recombinant protein structure, phosphorylation (regulates protein function), potency, and efficacy. It is crucial to study target proteins to establish whether the researchers isolated the correct recombinant protein.
Second, it also established whether all the recombinant proteins in a single batch feature similar characteristics. Similar to protein purification, recombinant protein isolation also requires specialized tools and techniques.
Recombinant protein production helps meet the demand for recombinant proteins for research in various fields. Therefore, following the above steps produces high-yielding target recombinant proteins that help improve or even save lives