Protein production

Source: Wikipedia, the free encyclopedia.
(Redirected from
Recombinant protein
)
This article is about the biotechnological method. For the natural process in cells, see Gene expression.
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

proteins and may be targeted to specific subcellular or extracellular locations.[1]

Protein production systems (also known as

biopharmaceuticals such as human insulin to treat diabetes, and to manufacture enzymes
.

Protein production systems

Commonly used protein production systems include those derived from

mammalian cells,[7][8] and more recently filamentous fungi such as Myceliophthora thermophila.[9] 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.[10]

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".[11][12] Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype.[13][14][clarification needed]

There are many ways to introduce foreign

posttranslational modification. Insect or mammal cell lines are used when human-like splicing of 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 difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding.

To address these concerns, expressions systems using multiple 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 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 production systems are developed using unicellular eukaryotes (i.e. non-pathogenic 'Leishmania' cells).

Bacterial systems

Escherichia coli
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.[3]

For example, a DNA sequence for a protein of interest could be

IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest.[2]

E. coli strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack lon and OmpT proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides T7 RNA polymerase (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead.[15]

Corynebacterium

Non-pathogenic species of the gram-positive

glutamate and lysine,[16]
components of human food, animal feed and pharmaceutical products.

Expression of functionally active human

secretory pathway (Sec) or the twin-arginine translocation pathway (Tat).[18]

Unlike

endotoxins in humans.[citation needed
]

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.[19]

Eukaryotic systems

Yeasts

Expression systems using either

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.[4][5]

Filamentous fungi

Filamentous fungi, especially Aspergillus and Trichoderma, have long been used to produce diverse industrial enzymes from their own genomes ("native", "homologous") and from recombinant DNA ("heterologous").[9]

More recently, Myceliophthora thermophila C1 has been developed into an expression platform for screening and production of native and heterologous proteins.The expression system C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media. C1 also does not "hyperglycosylate" heterologous proteins, as Aspergillus and Trichoderma tend to do.[9]

Baculovirus-infected cells
See also: BacMam

Baculovirus-infected insect cells[20] (Sf9, Sf21, High Five strains) or mammalian cells[21] (HeLa, HEK 293) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.[20][6] It is useful for production of proteins in high quantity. Genes are not expressed continuously because infected host cells eventually lyse and die during each infection cycle.[22]

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

Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells.[23][25] With this system, cells do not lyse and several cultivation modes can be used.[23] Additionally, protein production runs are reproducible.[23][24] This system gives a homogeneous product.[24] A drawback of this system is the requirement of an additional screening step for selecting viable clones.[25]

Excavata

Leishmania tarentolae (cannot infect mammals) expression systems 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.[citation needed]

Mammalian systems

The most common mammalian expression systems are Chinese Hamster ovary (CHO) and Human embryonic kidney (HEK) cells.[26][27][28]

Cell-free systems

Cell-free production 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.[29]

See also

References

  1. PMID 18235434
    .
  2. ^
    PMID 10508629
    .
  3. ^
    PMID 24860555
    .
  4. ^
    S2CID 35874864
    .
  5. ^
    PMID 21943899
    .
  6. ^
    PMID 15877075
    .
  7. PMID 15766864
    .
  8. PMID 18541133
    .
  9. ^
    doi:10.1089/ind.2011.7.214
    . Aspergillus and Trichoderma are currently the main fungal genera used to produce industrial enzymes.
  10. S2CID 22707536
    .
  11. ^ "Definition: expression system". Online Medical Dictionary. Centre for Cancer Education, University of Newcastle upon Tyne: Cancerweb. 1997-11-13. Retrieved 2008-06-10.
  12. ^ "Expression system - definition". Biology Online. Biology-Online.org. 2005-10-03. Retrieved 2008-06-10.
  13. ^ "overexpression". Oxford Living Dictionary. Oxford University Press. 2017. Archived from the original on February 10, 2018. Retrieved 18 May 2017. The production of abnormally large amounts of a substance which is coded for by a particular gene or group of genes; the appearance in the phenotype to an abnormally high degree of a character or effect attributed to a particular gene.
  14. ^ "overexpress". NCI Dictionary of Cancer Terms. National Cancer Institute at the National Institutes of Health. 2011-02-02. Retrieved 18 May 2017. overexpress
    In biology, to make too many copies of a protein or other substance. Overexpression of certain proteins or other substances may play a role in cancer development.
  15. PMID 19786035
    .
  16. PMID 19963020
    .
  17. PMID 16411922
    .
  18. S2CID 6238466
    .
  19. PMID 21968453
    .
  20. ^
    S2CID 34863069
    .
  21. PMID 10508635
    .
  22. PMID 16959350
    .
  23. ^
    doi:10.12665/j102.dyring
    .
  24. ^
    PMID 17046707
    .
  25. ^
    PMID 9353223
    .
  26. ^
    PMID 21968146
    .
  27. ^
    PMID 24721463
    .
  28. PMID 27322762
    .
  29. PMID 24161673
    .

Further reading

External links

Library resources about
Protein production
Retrieved from "https://en.wikipedia.org/w/index.php?title=Protein_production&oldid=1218480666"