What you Need to Know about Protein Production using Recombinant Technologies
Because of their important properties, proteins are usually utilized for nutritional, medical, and industrial applications.
21:08 09 June 2021
A key way to produce a large amount of a specific protein is called the recombinant DNA technology.
This involves using genetic recombination to bring genetic materials from several sources to create DNA sequences that are normally not found in the genome. And, proteins generated with the recombinant DNA technology are known as recombinant proteins. This post discusses what you need to know about protein production using recombinant technologies.
Recombinant protein production
Proteins play a huge part in life and they can manage all biochemical reactions and offer structure to organisms. Besides, proteins can act as transport for important molecules and use antibodies to defend the organism.
Depending on molecular biology’s central dogma, the blueprints for proteins are kept in DNA. You also need messenger RNA which is an intermediary template to transfer from the DNA the genetic information to the protein through the transcriptional and translational processes.
The first step involves protein expression that targets a large amount of proteins. This gene-to-protein process has two key steps called transcription and translation. It is usually achieved by manipulating gene expression in a particular organism so that it expresses a large amount of a recombinant gene. To get adequate amounts of the protein you desire, then codon optimization, strain selection, fusion systems, and many more techniques are used.
The next step is called protein purification that involves isolating the proteins from the samples like cell lysates or medium. Remember that protein refolding , chromatography, and cleavage of fusion moieties techniques can be used in protein purification processes.
Protein purification tends to focus on chromatographic purification of protein when it comes to research scale. Before you can identify and study properties of a specific protein, you need to separate and purify the protein. This uses several processes that intend to isolate a single or a couple of proteins from complex mixtures.
The purification process can separate the non-protein and protein parts of the mixture. Then you can separate the needed protein from the rest of the other proteins. This purification technique of a protein needs to be maximized to finish this process in a few steps.
Protein yield and activity may be optimized by choosing the proper lysis reagents and right purification resin. You can express many recombinant proteins as fusion proteins and have short affinity tags like glutathione S-transferase or polyhistidine, that allows selective purification of the desired protein.
No doubt, protein production can be a complex system in biotechnologies because each step may influence each other. The recombinant protein purification technique mostly depends on the properties of the recombinant protein as well as the expression system applied.
There are many firms out there that offer purified protein and gene services. That said, it’s worth noting that protein production refers to the biotechnological process you can use to produce a specific protein. Some of the commonly used production systems are insect cells, bacteria, yeast, mammalian cells, and many more.
It’s important to use the right expression system for specific applications if you want to achieve success. Protein functionality, solubility, yield, and purification speed are usually key factors you need to consider when selecting an expression system. Besides, each of these systems have their strengths and weaknesses, so it’s important to choose the right expression system.
Protein purification methods
Protein purification simply means protein fractionation and it’s also known as downstream processing. You need to remember that protein purification can involve several processes that includes pumping and ultrafiltration, and can involve a significant shear environment. Further, protein tags are a crucial and convenient way for streamlining protein purification, improving solubility of a recombinant protein, and allowing you to have a simple way to keep track of a protein during protein expression and purification.
There are a wide range of protein purification ways that you can combine to produce an ideal purification scheme. In most cases, you can use several purification steps and rarely, you can purify proteins in a single step.
The initial steps can combine both low-resolution as well as high-capacity techniques at later purification scheme’s stages. When it comes to low-resolution protein purification, techniques like fractional precipitation and even two-phase partition systems can be used.
For applications that need protein purification involving fairly small and high amounts of protein, you can choose chromatography to selectively purify the desired protein.
Key chromatography methods that you can use in protein purification include protein purification affinity chromatography, protein purification through ion exchange chromatography, and protein purification by HPLC.
With the protein production process, there are usually two steps. These are called transcription and translation and are called the central dogma in molecular biology. It means transcription and translation steps are part of the recombinant protein expression steps.
Therefore, to produce recombinant proteins, you need to isolate the gene and clone it into an expression system. Generating recombinant proteins needs the protein expression system alongside the protein purification as well as the protein identification systems.
There are several basic steps required to produce a recombinant protein. These include amplification of the gene, inserting into cloning vector, sub-cloning into expression vector, transforming into protein expressing system, testing to identify recombinant protein, large-scale production, isolation, and purification.
It’s also worth noting that there are many factors you need to consider. You need to figure out the ideal host system to select and the best way to isolate and purify a recombinant protein.
However, it can be hard to choose the right expression system or to utilize the proper purification method, especially when you have to consider the characteristics of the desired recombinant protein. Some of the things you need to consider include solubility, membrane bound, single or multi domain, size, and where to express it.
Keep in mind that recombinant protein production can be a time-consuming activity, especially if you don’t have enough experience needed to express and isolate recombinant proteins. If this is the case, then you need to contact a biological company that offers a protein expression service for different types of recombinant protein expression.
For example, biological companies can help you when it comes to transcription and translation.
Transcription happens in three steps in eukaryotes and prokaryotes, which are initiation, elongation, and termination. Keep in mind that transcription starts when you unwind the double-stranded DNA so that there can be binding of RNA polymerase.
When you initiate transcription, DNA releases RNA polymerase. Activators, repressors, and chromatin structures in eukaryotes can regulate transcription at various levels. In prokaryotes, there is no need for any special modification of messenger RNA and translation of the message begins before the transcription is done.
However, in eukaryotes, messenger RNA can be further processed to get rid of introns besides a cap at the 5’ end and several adenines at the messenger 3’ end to produce a polyA tail. Then a modified messenger RNA can be transferred to the cytoplasm for translation.
Protein synthesis or translation refers to a multi-step process that needs macromolecules, such as ribosomes, protein factors, transfer RNAs, small molecules like ATP and amino acids. Remember that there are particular protein factors required for each step of translation. The entire process is quite similar in both eukaryotes and prokaryotes, though there are some differences.
During the initiation, there is a small subunit of the ribosome that binds to the initiator t-RNA and scans the messenger RNA beginning at the 5’ end. It does this to identify and bind an initiation codon. On the other hand, the large subunit of the ribosome tends to join the small ribosomal subunit to produce the initiation complex that is at the initiation codon.
Protein factors and sequences in messenger RNA are usually involved when it comes to recognizing the initiation codon as well as the creation of the initiation complex. In elongation step, tRNAs can bind to the designed amino acids, the process called tRNA charging and move them to the ribosome to be polymerized and form a peptide.
The amino acids sequence that you add to the growing peptide depends on the messenger RNA sequence of the transcript. Lastly, the nascent polypeptide is eventually released during the termination step once the ribosome gets to the termination codon. So at this stage, the ribosome gets released from the messenger RNA and can initiate another process of translation.
After translation, polypeptides can be modified in several ways to finish their structure. Also, they can designate their place or regulate their activities in the cell. A post-translational modification refers to the multiple additions or changes to the chemical structure and are important features of the entire cell biology.
Forms of post-translational modifications can include polypeptide folding into globular proteins with the assistance of chaperone proteins to get to the low energy state and modifications of the present amino acids. Others are disulfide bridge reduction or formation, protein modifications that help binding functions, and addition of functional groups to regulate protein activity.
The uses of recombinant proteins
Recombinant proteins are mainly used in biomedical research to help experts understand health and disease issues. Simply put, recombinant proteins can be necessary tools you can use to understand protein-protein interactions. Protein interactions are usually characterized as transient or stable and play a huge role in a cellular process.
Recently, RP microarrays used for checking protein to protein interactions are becoming quite popular. With this approach, many researchers usually seed a slide with many immobilized proteins. They then treat with various molecules to check how the two agents are interacting with each other. Using this system, researchers have studied how proteins interact with other peptides or proteins, small molecules, enzymes, nucleic acids, and lipids. This allows high throughput when you are studying protein to protein interactions.
Even better, recombinant proteins have been effective in many laboratory techniques. Recombinant proteins are usually used to generate enzymatic assays. Therefore, when you use them together with a matched antibody pair, you can utilize recombinant proteins as standards and positive controls in various laboratory techniques.
Recombinant proteins can also serve as great tools for examining cellular response associated with stress, and disease situations. Peptides and recombinant proteins can be given to animal models of disease to help researchers in identifying therapeutic potentials.
Recombinant proteins also come in handy in biotherapeutics. Many human diseases are partially or systematically associated with dysfunction of certain proteins. Therapeutic proteins offer crucial therapies for several diseases like cancer, diabetes, hemophilia, infectious diseases, and anemia.
Some of the common therapeutic proteins are antibodies, hormones, enzymes, interleukins, and anticoagulants. But human proteins that are obtained via genetic engineering can play a crucial role when it comes to the therapeutic medicines market. For example, one of the RP vaccines that was approved by the FDA is called the Hepatitis B vaccine designed to prevent infection that is caused by subtypes of the Hepatitis B virus.
The first recombinant protein utilized in treatment was perhaps recombinant human insulin that was developed in 1982. Since then, the recombinant protein industry has grown significantly. Today, there are more than 100 recombinant proteins that have been approved by the United States FDA for clinical use.
But there are more recombinant proteins that are generated and utilized in medicine worldwide. Also, recombinant human insulin was an early example of how biotechnology can be used in drug development. Recombinant proteins are mostly potent medicines that can be safe to use and take a shorter period to generate than small molecules.
Some of the recombinant proteins utilized in the clinic are recombinant hormones, growth factors, blood clotting factors, interleukins, interferons, enzymes, thrombolytic drugs, and many more. It’s worth noting that these recombinant proteins can treat various diseases including congestive heart failure, diabetes, asthma, sclerosis, cancers, rheumatoid arthritis and dwarfism.
Recombinant proteins can also be used in agriculture, food production, and bioengineering. For instance, in the breeding industry, you can add enzymes to animal feed to improve the nutritional value, reduce feed costs, and enhance animal performance. Above all, lactic acid bacteria have been utilized for years to produce fermented foods.