Recombinant proteins are found predominantly in pharmaceutical and biotechnology industries, but they are found in detergents, cosmetics, paper and used for biological research studies . rDNA containing a cloned gene is introduced into cells or organisms where the cloned gene is transcribed, making an mRNA copy, which is translated into the amino acid sequence of the protein. Diabetes has been treated with insulin since the 1920s but before recombinant insulin was licensed as a drug in 1982, it was purified from cow or pig pancreas . Today recombinant insulin is produced in Escherichia coli bacteria or yeast cells . Cheesemaking uses enzymes collectively called rennet used to clot milk . Chymosin, also known as rennin, is one of these milk-clotting enzymes which used to be extracted from calf stomachs and is now produced as a recombinant protein.
The most common organisms used to express recombinant proteins for pharmaceutical use are bacteria, yeast and mammalian cell cultures. Microbial expression systems like bacteria and yeast are faster and cheaper than mammalian systems because microbial cultures grow faster, and growth media costs 90% less than cell culture media used for growing Chinese hamster ovary (CHO). Mammalian cells are more sensitive to culture conditions . E. coli and S. cerevisiae are the most popular bacteria and yeast used for recombinant protein production because they have been such widely used model organisms with plenty of synthetic biology tools available . The most common mammalian cells used to produce recombinant proteins are Chinese hamster ovary and murine myeloma cell lines .
Yeast and mammalian cells are eukaryotic cells while bacteria are prokaryotic cells. Differences between the two cell types, such as the presence of a nucleus and membrane-bound organelles in eukaryotes that are absent in prokaryotes, affect how proteins are processed. Eukaryotic cells can express larger proteins, like monoclonal antibodies which are composed of four subunits, and they secrete fully assembled protein complexes . Multidomain proteins expressed in E.coli often lack activity .
Different cell types add different types of protein modifications after the protein is translated, like glycosylation. Glycosylation and other post-translational modifications can be added as in vitro processing steps. Glycosylation in mammalian cell lines are most similar to humans. However, some non-human post-translational modifications produced in mammalian cells can potentially be immunogenic . Approved recombinant therapeutic products have been produced in human embryonic kidney 293 (HEK293) and fibrosarcoma HT-1080 cell lines.
Yeast and mammals can secrete active proteins in their culture medium but bacteria like E. coli accumulate protein internally and the protein may be insoluble. From E. coli, cell lysis is needed to harvest the target protein, a process that also releases proteases and endotoxins, enzymes and molecules that can degrade or contaminate the protein product, and insoluble proteins require additional processing steps . Insolubility is a major reason why pharmaceutical proteins are terminated early in clinical development.
A strain of gram-positive Corynebacterium glutamicum bacteria does not produce endotoxins and has been engineered to secrete active proteins into the culture media. C. glutamicum has been used to produce both eukaryotic and prokaryotic proteins including human epidermal growth factor and green fluorescent protein (GFP) . An expression system based on C. glutamicum called Corynex by Japanese company, Ajinomoto Co., Inc..
Insulin and glucagon are produced industrially in Saccharomyces cerevisiae yeast cultures and are used as drugs to regulate sugar levels in people with Diabetes . Recombinant human insulin is also produced in the yeast species Komagatella phaffii (previously classified as Pichia pastoris), which is also used for other recombinant proteins like human serum albumin, hepatitis B vaccine, interferon-alpha 2b, trypsin and collagen . Kluyveromyces lactis is known in the food industry for secreting beta-galactosidase which is used for making lactose-free products, but it is used to make recombinant proteins for food and pharmaceutical applications.
Transgenic plants and non-transgenic plants are used to produce recombinant proteins. Transgenic plants can be made by introducing rDNA into the plant by Agrobacterium mediated transformation or by particle bombardment with a gene gun. Non-transgenic plants like tobacco and cowpeas can be infected with recombinant tobacco mosaic virus or recombinant cowpeas mosaic virus to introduce the gene to be produced. Recombinant phytase, an enzyme used in animal feed, can be produced by introducing microbial phytase genes into plants like tobacco, canola and soybean . Hirudin, an anticoagulant protein used to treat thrombosis was originally isolated from the leech Hirudo medicinalis and is produced in bacteria and yeast, but has been shown to be produced in transgenic plants. A human therapeutic protein, human somatotropin was produced in tobacco leaves after the rDNA was introduced and integrated into chloroplasts DNA in tobacco leaves by particle bombardment.
The first recombinant protein drug made in plants on the market in 2012 was produced in cultured carrot cells. Elelyso (taliglucerase alfa) treats symptoms of the lysosomal storage disorder Gaucher disease .
Plants have less risk of contamination with pathogenic microbes compared with mammalian cell or microbial cell expression systems. Plant cultivation is less complex and less expensive than ell culture and plant proteins can be expressed in the edible parts of the plants, which for some products could eliminate the need for purification. On the downside there is the possibility of contamination of plant produced recombinant proteins with herbicides and pesticides and outdoor plant growth is seasonal . For therapeutic proteins plant expression systems need to overcome limitations like low level expression and post-translational modifications of the recombinant proteins are much different in plants compared to mammalian cells.
Documentaries, videos and podcasts
- Asilomar Conference on Recombinant DNA
- Recombinant DNARecombinant DNA (rDNA) is the combining of DNA from different organisms. rDNA technology allows the cloning of genes and genomic elements to study their function. Genetic engineering uses knowledge about genetic elements and rDNA technology tools to construct rDNA for research, medicine and food applications.
- Cellular agricultureCellular agriculture is an interdisciplinary scientific field drawing from several disciplines such as synthetic biology, genetic engineering, molecular biology, tissue engineering, biochemistry, and food science to design organisms capable of producing a wide variety of agricultural products.