Proteins are extensively employed in industrial, nutritional, and medicinal applications due to their essential functions. Recombinant DNA technology, which involves leveraging genetic recombination to link together genetic material from diverse sources, generating DNA sequences that are not typically found in the genome, is a fundamental approach to manufacture vast amounts of a specific protein. In more simple terms, recombinant proteins are proteins that have been created using recombinant DNA technology.
This guide will discuss in detail the following topics to help you better understand how recombinant custom proteins expression work:
- What are recombinant DNAs?
- What is the best way to synthesise recombinant DNA?
- What is a recombinant protein, and how does it work?
- What is the best way to produce a recombinant protein?
Let’s dive right in.
What Are Recombinant DNAs?
To understand recombinant DNA and how to use recombinant DNA technology to make a specific protein, you must first understand how proteins are expressed inside an organism. Proteins are one of the most significant components that make up an organism’s body and conduct vital activities. For example, digestive enzymes secreted by your digestive tract break down macromolecules in food into smaller molecules that can be absorbed by the body, allowing you to digest them. These enzymes are all proteins.
Genetic concepts have been intensively investigated since the present structure of DNA was discovered in 1953. Proteins are now widely understood as generated from DNA in a two-step process. The first stage is transcription, which involves making mRNA from DNA. The next stage is translation, which consists of protein production from mRNA. A DNA sequence, in other words, contains instructions for producing a certain protein. As a result, gene expression and protein expression are interchangeable terms.
Recombinant DNA (rDNA) is a DNA strand made up of two or more DNA sequences that have been combined. Recombination of genes is a natural occurrence. The so-called recombinant DNA technology allows the process to be purposefully controlled for numerous reasons. Using recombinant DNA technology, scientists can build novel sequences that do not naturally arise under standard settings and environmental conditions.
The recombinant DNA is brought into a host organism translated into a new protein known as a recombinant protein. The manufacturing of recombinant proteins for pharmaceutical, medicinal, agricultural, and other purposes relies heavily on recombinant DNA technology.
What is The Best Way to Synthesise Recombinant DNA?
Gene, DNA, and molecular cloning are all terms used to describe recombinant DNA technology. They all relate to the introduction of foreign DNA into a genetic element of a host organism that’s self-replicating, which results in the foreign DNA’s amplification. There are three primary approaches used in creating recombinant DNA at the moment, namely:
Transformation of Non-Bacterial Organisms
In this method, bacteria are not used as a host cell in the production process. DNA microinjection, for example, is a technique in which foreign DNA is directly injected into the nucleus of a host cell. The process is called biolistic. Biolistics is a technique for bombarding foreign DNA into a target cell using high-velocity microprojectiles.
Transformation
A segment of foreign DNA is cut and put into a vector, most often a plasmid. The resultant vector is then inserted into a host cell, such as E. coli to express the foreign DNA segment. Hence, Transformation is the process that involves a bacterial cell absorbing foreign DNA.
Phage Introduction
A phage is used to introduce foreign DNA into a target cell, with the phage DNA containing the foreign DNA is subsequently integrated into the genome of the host cell.
What is Recombinant Protein, And How Does it Work?
Recombinant proteins are proteins that have been created artificially using recombinant DNA technology. Diagnostic instruments, vaccinations, medicines, detergents, cosmetics, food manufacturing, and feed additives are just a few applications for proteins. The increased demand for proteins cannot be met by simply extracting proteins from their natural sources. Recombinant DNA technology makes it easier to obtain vast quantities of proteins.
This strategy has both benefits and drawbacks. Insulin, for example, a hormone that regulates blood sugar and is lowered in diabetic patients, has already been manufactured using recombinant DNA technology, saving countless lives. Furthermore, recombinant DNA technology allows for the customization of the characteristics of the desired protein.
What is the best way to produce a recombinant protein?
Scientists used to be the only ones who could make recombinant proteins. However, recombinant protein synthesis has evolved into a mature and widely used technology thanks to the development of simple, commercially available technologies. One of the major challenges in the recombinant protein synthesis process is that you will be confronted with a dizzying selection of options.
- Which system should the protein be expressed in?
- What is the best expression vector to use?
- Can the protein expression be in its whole or parts?
- Is it necessary to tag the protein?
- What is the best approach for purifying the protein
When making recombinant proteins, you’ll have to consider several decisions. If you make the right choices, you’ll get high-quality recombinant proteins and more successful follow-up tests. However, if you make the wrong judgement, you may not get the recombinant protein you require, or the recombinant protein’s quality and purity may fall short of your expectations.
Furthermore, because each protein is unique, there is no one-size-fits-all solution. The protein you want to express has a significant role in deciding which of the several manufacturing techniques to use.
Several proteins from diverse species (bacteria, viruses, Archaea, and Eukarya) have been created and purified in the lab during the last several decades using recombinant DNA technology. The following are some of the major phases in the creation of recombinant proteins, as summarised by several researchers:
- Making the expression clone after getting the cDNA
- Cloning
- Creating an appropriate system for protein expression
- Expression test on a minor scale
- Purification of proteins
- Characterization of proteins
Several techniques for expressing recombinant proteins are now available, encompassing both cell-based and cell-free approaches. Cell-based systems can be further classified into eukaryotic and prokaryotic systems. A variety of variables influences the expression of recombinant proteins. For example, researchers typically require high protein production yields; however, inclusion bodies may form if the recombinant protein is produced too quickly. Hence, many recombinant proteins require modifications such as glycosylation, only obtainable in eukaryotic cells, so prokaryotic cells such as E. coli are not suitable for protein expression in this case.
Conclusion
Recombinant protein manufacturing is a well-established technology with several commercially accessible systems. However, there are still difficulties in manufacturing and purifying recombinant proteins.
