alt=Central dogma depicting transcription from DNA code to RNA code to the proteins in the second step covering the production of protein.|thumb|[[Central dogma of biology|Central dogma depicting transcription from DNA code to RNA code to the proteins in the second step covering the production of protein.]]
Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.
Protein production systems (also known as expression systems) are used in the life sciences, biotechnology, and medicine. Molecular biology research uses numerous proteins and enzymes, many of which are from expression systems; particularly DNA polymerase for PCR, reverse transcriptase for RNA analysis, restriction endonucleases for cloning, and to make proteins that are screened in drug discovery as biological targets or as potential drugs themselves. There are also significant applications for expression systems in industrial fermentation, notably the production of biopharmaceuticals such as human insulin to treat diabetes, and to manufacture enzymes.
Protein production systems
Commonly used protein production systems include those derived from bacteria, yeast, baculovirus/insect, mammalian cells, and more recently filamentous fungi such as Myceliophthora thermophila. When biopharmaceuticals are produced with one of these systems, process-related impurities termed host cell proteins also arrive in the final product in trace amounts.
Cell-based systems
The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level". Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype.
There are many ways to introduce foreign DNA to a cell for expression, and many different host cells may be used for expression — each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the host and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria (such as E. coli, B. subtilis), yeast (such as S. cerevisiae
Corynebacterium
Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine, components of human food, animal feed and pharmaceutical products.
Expression of functionally active human epidermal growth factor has been done in C. glutamicum, thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general, secretory pathway (Sec) or the twin-arginine translocation pathway (Tat).
Unlike gram-negative bacteria, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.
Pseudomonas fluorescens
The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein.
Eukaryotic systems
Yeasts
Expression systems using either S. cerevisiae or Pichia pastoris allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media of proteins. (Sf9, Sf21, High Five strains) or mammalian cells (HeLa, HEK 293) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.
Non-lytic insect cell expression
Non-lytic insect cell expression is an alternative to the lytic baculovirus expression system. In non-lytic expression, vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent gene expression. This is followed by selection and screening of recombinant clones. The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression.
- Chinese hamster ovary cell
See also
- Cellosaurus, a database of cell lines
- Gene expression
- Single-cell protein
- Protein purification
- Precision fermentation
- Host cell protein
- List of recombinant proteins