Protein expression (biotechnology)

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Protein production can occur after gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins.[citation needed] Protein expression is commonly used by proteomics researchers to denote the measurement of the presence and abundance of one or more proteins in a particular cell or tissue.

Protein production systems (in lab jargon also referred to as "expression systems") are very widely 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 and restriction endonucleases for cloning. There are also significant medical applications for expression systems, notably the production of human insulin to treat diabetes. Protein expression systems are used to produce certain proteins in biotechnology and industry, and more recently to produce sets (combinatorial series) of protein that are screened for drug discovery purpose.

Protein production systems[edit]

Commonly used protein expression systems include those derived from bacteria,[1] yeast,[2][3]baculovirus/insect,[4] and mammalian cells.[5][6] and more recently filamentous fungi such as the commercially relevant fungus Myceliophthora thermophila[1]

Cell-based systems[edit]

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".[7][8] Expression is often done to a very high level and therefore referred to as overexpression.

There are many ways to introduce foreign DNA to a cell for expression, and there are many different host cells which 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[3]) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses (such as baculovirus, retrovirus, adenovirus), plasmids, artificial chromosomes and bacteriophage (such as lambda). The best expression system of choice depends on the gene involved, for example the Saccharomyces cerevisiae is often preferred for proteins that require significant posttranslational modification and Insect or mammal cell lines are used when human-like splicing of the mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.

Because bacteria are prokaryotes, they are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. Hence, multi-domain eukaryotic proteins expressed in bacteria often are non-functional. Also, many proteins become insoluble as inclusion bodies that are very difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding procedures.

To address theses concerns, expressions systems using several eukaryotic cells were developed for applications requiring the proteins be conformed as in, or closer to eukaryotic organisms: cells of plants (i.e. tobacco), of insects () or mammalians (i.e. bovines) are transfected with genes and cultured in suspension and even as tissues or whole organisms, to produce fully folded proteins. Mammalian in vivo expression systems have however low yield and other eventual limitations (time-consuming, toxicity to host cells,..). To combine the high yield/productivity and scalable protein features of bacteria and yeast, and advanced epigenetic features of plants, insects and mammalians systems, other protein expression systems are developed using unicellular eukaryotes (i.e. non-pathogenic 'Leishamania' cells).

Bacterial systems[edit]

Escherichia coli[edit]
E. coli, one of the most popular hosts for artificial gene expression.

E. coli is one of the most widely used expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription.

For example a DNA sequence for a protein of interest could be cloned or subcloned into a high copy-number plasmid containing the lac promoter, which is then transformed into the bacterium Escherichia coli. Addition of IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest.

Corynebacterium[edit]

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,[9] components of human food, animal feed, and pharmaceutical products.

Expression of functionally active human epidermal growth factor has been done in C. glutamicum,[10] 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).[11]

Unlike gram-negative bacteria, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.

Pseudomonas fluorescens[edit]

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.[12]

Eukaryotic systems[edit]

Saccharomyces cerevisiae, Pichia Pastoris[edit]

Expression systems in yeast typically use the common and well known S.cerevisiae, but also Bacillus gender. Systems using Pichia pastoris allow stable and lasting production of proteins closer to mammalian cells, at high yield, in chemically defined media of proteins.

Filamentous fungi[edit]

Filamentous fungi, especially Aspergillus and Trichoderma, but also more recently Myceliophthora thermophila, C1 [2] have been developed into expression platforms for screening and production of diverse industrial enzymes. C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media.

Baculovirus-infected cells[edit]

Infected insect cells[13] (Sf9, Sf21, High Five strains) or mammalian cells[14] (HeLa, HEK 293) allows expression of glycosylated proteins that cannot be expressed using yeast or prokaryotic cells (like E. coli). It is very useful system for expression of proteins in high quantity. Genes are not expressed continuously because infected host cells will eventually lyse and die during each infection cycle.[15]

Non-lytic insect cell expression[edit]

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 protein expression.[16][17] This is followed by selection and screening of recombinant clones.[18] The non-lytic system has been used to give higher protein yield and quicker expression of recombinant proteins compared to baculovirus-infected cell expression.[17] Cell lines used for this system include: Sf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells.[16][18] With this system, cells do not lyse and several cultivation modes can be used.[16] Additionally, protein production runs are reproducible.[16][17] This system gives a homogeneous product.[17] A drawback of this system is the requirement of an additional screening step of selecting viable clones.[18]

Leishamania[edit]

Protozoan Leishmania tarentolae (non pathogenic strain) expression system, also licensed,[19] allow stable and lasting production of proteins at high yield, in chemically defined media. Produced proteins exhibit fully eukaryotic post-translational modifications, including glycosylation and disulfide bond formation.

Plant systems[edit]

Tobacco

Mammalian systems[edit]

Bos primigenius (Bovine)
Mus musculus (Mouse)
Chinese Hamster Ovary
Human Embryonic Kidney cells
Baby Hamster Kidney

Cell-free systems[edit]

Cell-free expression of proteins is performed in vitro using purified RNA polymerase, ribosomes, tRNA and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system. Due to the low expression levels and high cost of cell-free systems cell-based systems are more widely used.[citation needed]

See also[edit]

References[edit]

  1. ^ Baneyx F (October 1999). "Recombinant protein expression in Escherichia coli". Curr. Opin. Biotechnol. 10 (5): 411–21. doi:10.1016/s0958-1669(99)00003-8. PMID 10508629. 
  2. ^ Cregg JM, Cereghino JL, Shi J, Higgins DR (September 2000). "Recombinant protein expression in Pichia pastoris". Mol. Biotechnol. 16 (1): 23–52. doi:10.1385/MB:16:1:23. PMID 11098467. 
  3. ^ a b Malys N, Wishart JA, Oliver SG, McCarthy JE (2011). "Protein production in Saccharomyces cerevisiae for systems biology studies". Methods Enzymol. 500: 197–212. doi:10.1016/B978-0-12-385118-5.00011-6. PMID 21943899. 
  4. ^ Kost TA, Condreay JP, Jarvis DL (May 2005). "Baculovirus as versatile vectors for protein expression in insect and mammalian cells". Nat. Biotechnol. 23 (5): 567–75. doi:10.1038/nbt1095. PMID 15877075. 
  5. ^ Rosser MP, Xia W, Hartsell S, McCaman M, Zhu Y, Wang S, Harvey S, Bringmann P, Cobb RR (April 2005). "Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system". Protein Expr. Purif. 40 (2): 237–43. doi:10.1016/j.pep.2004.07.015. PMID 15766864. 
  6. ^ Lackner A, Genta K, Koppensteiner H, Herbacek I, Holzmann K, Spiegl-Kreinecker S, Berger W, Grusch M (September 2008). "A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells". Anal. Biochem. 380 (1): 146–8. doi:10.1016/j.ab.2008.05.020. PMID 18541133. 
  7. ^ "Definition: expression system". Online Medical Dictionary. Centre for Cancer Education, University of Newcastle upon Tyne: Cancerweb. 1997-11-13. Retrieved 2008-06-10. 
  8. ^ "Expression system - definition". Biology Online. Biology-Online.org. 2005-10-03. Retrieved 2008-06-10. 
  9. ^ Brinkrolf K, Schröder J, Pühler A, Tauch A (September 2010). “The transcriptional regulatory repertoire of Corynebacterium glutamicum: reconstruction of the network controlling pathways involved in lysine and glutamate production.” J Biotechnol. 149 (3): 173-82.
  10. ^ Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi Y (January 2006). “Secretion of human epidermal growth factor by Corynebacterium glutamicum.” Lett Appl Microbiol. 42 (1): 66-70.
  11. ^ Meissner D, Vollstedt A, van Dijl JM, Freudl R (September 2007). “Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria.” Appl Microbiol Biotechnol. 76 (3): 633-42.
  12. ^ Retallack, Jin, Chew (2011). "Reliable Protein Production in a Pseudomonas fluorescens Expression System." Protein Expression and Purification 81 (2011): 157-65.)
  13. ^ Altmann, Friedrich; Staudacher, E; Wilson, IB; März, L (1999). "Insect cells as hosts for the expression of recombinant glycoproteins". Glycoconjugate Journal 16 (2): 109–23. doi:10.1023/A:1026488408951. PMID 10612411. 
  14. ^ Kost, T; Condreay, JP (1999). "Recombinant baculoviruses as expression vectors for insect and mammalian cells". Current Opinion in Biotechnology 10 (5): 428–33. doi:10.1016/S0958-1669(99)00005-1. PMID 10508635. 
  15. ^ Yin, Jiechao, Guangxing Li, Xiaofeng Rena, and Georg Herrler. “Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes.” Journal of Biotechnology 127 (2007) 335–347.
  16. ^ a b c d Dyring, Charlotte. “Optimising the drosophila S2 expression system for production of therapeutic vaccines”. Bioprocessing Journal 10 (2011) 28-35.
  17. ^ a b c d Olczak, Mariusz and Teresa Olczak. “Comparison of different signal peptides for protein secretion in nonlytic insect cell system.” Analytical Biochemistry 359 (2006) 45-53.
  18. ^ a b c McCarroll, L. and L.A. King. “Stable insect culture for recombinant protein production.” Current Opinions in Biotechnology 8 (1997) 590-594.
  19. ^ http://www.jenabioscience.com/cms/en/1/browse/1007_eukaryotic_expression_system_lexsy.html Jena Biosciences

Further reading[edit]

  • Higgins SJ, Hames BD. Protein Expression: A Practical Approach. Oxford, UK: Oxford University Press. p. 304. ISBN 0-19-963623-0. 
  • Baneyx F (2004). Protein Expression Technologies: Current Status and Future Trends. Norfolk, UK: Horizon Bioscience. p. 548. ISBN 0-9545232-5-3. 

External links[edit]