Recombinant protein is a protein coded by the manipulated gene - Recombinant Gene and generated by the specific recombinant expression system (see protein expression systems). The recombinant gene is a new genetic combination that one or more DNA segments or genes have been inserted from a different molecule or from from other species. Compared with natural protein, recombinant protein can be produced in large amounts with relative ease.
Recombinant protein plays an important role in studying biological processes such as Cytokines and Growth Factors, enzymes and kinases researches and complement system functions. Moreover, recombinant proteins are known as highly potent medicines that are safe from off-target side effects, take a shorter time to develop than small molecules (see recombinant protein drugs).
Among the biological study and protein functions researchment, one of the most common methods is recombinant fusion protein, or fusion protein for short. What is recombinant fusion protein?
A recombinant fusion protein is created through the combination of two or more genes that originally coded for separate proteins. Translated from the fusion gene, the recombinant fusion protein containing a single or multiple polypeptides gains functional properties derived from each of the original proteins.
What are uses of recombinant fusion proteins? There are three most important uses of fusion proteins:
• as aids in the purification of cloned genes
• as reporters of expression level
• as histochemical tags to enable visualization of the location of proteins in a cell, tissue, or organism
In recombinant protein purification, recombinant fusion protein method is commonly used in affinity chromatography where a protein that can be easily and conveniently purified is linked to a protein that the researcher wishes to study. A number of proteins and peptides have been used as fusion tags, including glutathione-S-transferase (gst tag), maltose-binding protein (mbp tag), polyhistidine (his tag), human growth hormone, ubiquitin, and antibody epitopes. In addition, β-galactosidase, luciferase, and green fluorescent protein (GFP) are usually linked as reporters of the parter proteins expression and location.
Over 180 recombinant protein drugs are in use as therapeutic agents today. Therapeutic recombinant protein drugs are an important class of medicines serving patients most in need of novel therapies. Having revolutionized therapy options in many disease areas, recombinant protein drugs are now a important component of the overall health-care industry. What is therapeutic function of recombinant protein drugs? Nearly half of therapeutic recombinant proteins are monoclonal antibodies, the others can be subdivided by function as follows:
• Replacements for proteins that are missing or defective
• Increasing the amount of proteins already present
• Inhibition of infectious agents, such as bacteria or viruses
• Carriers for other molecules (mostly still in development)
Some examples of recombinant proteins drugs are listed below:
|Erythropoietin||Promoting red blood cell formation in the treatment of anemia|
|Factor VIII||Helping blood clots form in hemophiliacs|
|Filgrastim and sargramostim (blood cell–stimulating bone marrow factors)||Boosting white blood cell counts after radiation therapy or transplantation|
|Insulin-like growth factor 1 (IGF1)||Treating certain growth problems|
|Interferon (alpha)||Treating hepatitis B and C, genital warts, certain leukemias and other cancers|
|Interferon (beta)||Treating multiple sclerosis|
|Interferon (gamma)||Treating chronic granulomatous disease|
|Interleukin-2||Killing tumor cells|
|Somatotropin||Treating growth hormone deficiency|
|Tissue plasminogen activator (t-PA)||Dissolving blood clots to prevent heart attacks and lessen their severity|
Recombinant proteins provided important breakthroughs in biomedical biotechnology, especially in human therapeutic research. Most recombinant proteins in therapeutic use are from humans but are expressed in other organisms such as bacteria, yeast, or animal cells in culture. In addition, human genes often contain large introns. Therefore, an intron-free version of the gene is often made by converting the mRNA into cDNA.
The first human recombinant protein used in treatment is recombinant human insulin developed in 1982. Then the recombinant protein industry has rapidly grown and there are many types of therapeutic recombinant proteins forming a comprehensive system of recombinant human proteins.
To make recombinant proteins, the gene is isolated and cloned into an expression vector. Generating a recombinant protein requires the protein expression system, protein purification system and protein identification systems.
Basic steps to get recombinant Protein:
1. Amplification of gene of interest.
2. Insert into cloning vector.
3. Sub cloning into expression vector.
4. Transformation into protein expressing host (bacteria (E coli), yeast, mammalian cells or baculovirus-insect cell system).
5. Test for identification of recombinant protein.( Western blot or Fluoroscence)
6. Large scale production. (Large scale fermentor)
7. Isolation and purification.
Recombinant proteins are clinically relevant proteins produced in large scale. The gene for the protein of interest is cloned into a vector and expressed into protein in a model organism. Recombinant protein production requires a system technologies of recombinant gene technology, protein expression and purification technology. Sino Biological provides professional recombinant protein services with years of experience for about 6000 recombinant proteins production.
• DNA Isolation and Purification: DNA can be isolated by first removing the cell wall and cell membrane components. Next, the proteins are removed by phenol, and finally, the RNA is removed by ribonuclease.
• Protein Expression: In order to avoid toxic effects of protein overproduction and insure protein refold in right way, increase protein stability and improving protein secretion, it is important to select the right vectors and the best expression system among mammalian cells, insect cells, yeast, and bacteria. Each recombinant protein must be evaluated for which system will work the best.
• Protein Purification: The purpose of protein purification is to separate the target protein from all components of the cell division fluid while still retaining the biological activity and chemical integrity of the protein. The separation and purification of proteins is an arduous and arduous task, and it is necessary to select a suitable purification method according to the characteristics of the protein to improve the purity of the obtained protein product.
What is recombinant DNA mean? Recombinant DNA, or recombinant gene technology is a method where genes, the DNA molecules that containing genetic information are manually manipulated for a certain purpose and programme, recombined with genes of the same source or heterogeneous origin, and then introduced into appropriate receptor cells, where the recombinant DNA (gene) is amplified and expressed at the same time.
Basic to all biotechnology research is the ability to manipulate DNA, containing DNA isolation and purification, electrophoresis separation of DNA fragments, restriction enzymes and ligase, radioactive labeling of nucleic acids and autoradiography, fluorescence detection, Southern and Northern blots, Fluorescence In Situ Hybridization (FISH) ,and gene library construction technology.
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Assenberg R, et al. (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23(3): 393-402.
Ohba Y, et al. (2013) Fluorescent protein-based biosensors and their clinical applications. Prog Mol Biol Transl Sci 113: 313-348.