Pichia pastoris
Encyclopedia
Pichia pastoris is a species of methylotroph
ic yeast
. Pichia is widely used for protein expression
using recombinant DNA
techniques. Hence it is used in biochemical
and genetic
research in academia
and the biotechnical industry
.
Pichia pastoris has two alcohol oxidase
genes, AOX1 and AOX2, which have a strongly inducible
promoter. These genes allow Pichia to use methanol as a carbon
and energy source. The AOX promoters are induced by methanol
and are repressed by e.g. glucose
. Usually the gene for the desired protein is introduced under the control of the AOX1 promoter, which means that protein production can be induced by the addition of methanol. In a popular expression vector, the desired protein is produced as a fusion product to the secretion
signal of the α-mating factor from Saccharomyces cerevisiae
(baker's yeast). This causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification
. There are commercially available plasmid
s that have these features incorporated (such as the pPICZα vector).
research, the bacterium Escherichia coli
is the most frequently used organism for production of recombinant DNA
and proteins. This is due to E. coli's fast growth rate, good protein production rate and undemanding growth conditions. Protein expression in E. coli is usually faster than in Pichia pastoris for several reasons: Competent E. coli cells can be stored frozen, and thawed immediately before use, whereas Pichia cells have to be produced immediately before use. Expression yields in Pichia vary between different clones
, and usually a large number of clones needs to be screened for protein expression before a good producer is found. Optimal induction times of Pichia are usually on the order of days, whereas E. coli usually reaches optimal yields within hours of induction. The major advantage of Pichia over E. coli is that Pichia is capable of producing disulfide
bonds and glycosylation
s in proteins. This means that in cases where disulfides are necessary, E. coli might produce a misfolded protein, that is usually inactive or insoluble.
The well-studied Saccharomyces cerevisiae
(baker's yeast) is also used as an expression system with similar advantages over E. coli as Pichia. However Pichia has two main advantages over S. cerevisiae in laboratory and industrial settings:
Firstly, Pichia, as mentioned above, is a methylotroph
, meaning it can grow with the simple alcohol
methanol
as its only source of energy — Pichia can easily be grown in cell suspension in reasonably strong methanol solutions that would kill most other micro-organisms, a system that is cheap to set up and maintain.
Secondly, Pichia can grow to very high cell densities, and under ideal conditions can multiply to the point where the cell suspension is practically a paste. As the protein yield from expression in a microbe is roughly equal to the product of the protein produced per cell and the number of cells, this makes Pichia of great use when trying to produce large quantities of protein without expensive equipment.
Compared to other expression systems such as S2-cells from Drosophila melanogaster
or Chinese Hamster Ovary cell
s, Pichia usually gives much better yields. Cell lines from multicellular organisms usually require complex rich media, including amino acids, vitamin
s and growth factor
s. These media significantly increase the cost of producing recombinant proteins. Additionally, since Pichia can grow in media containing only one carbon source and one nitrogen
source, it is suitable for isotopic labelling applications in e.g. protein NMR. However, a number of proteins require chaperonin
s for proper folding. Thus, Pichia is unable to produce a number of proteins for which the host lacks the appropriate chaperones. In 2006 a research group has managed to create a strain that produces Erythropoietin
in its normal glycosylation form. This was achieved by exchanging the enzymes responsible for the fungal type glycosylation, with the mammalian homologs. Thus, the altered glycosylation pattern allowed the protein to be fully functional.
(also known as baker's yeast). As a model organism
for biology, and having been used by man for various purposes throughout history, S. cerevisiae is well studied, to say the least. The two yeast species (Pichia, Saccharomyces) have similar growth conditions and tolerances, and thus the culturing of Pichia pastoris can be readily adopted by labs without specialist equipment.
The Pichia pastoris GS115 genome has been sequenced by the Flanders Institute for Biotechnology (VIB) and Ghent University (UGent) and published in Nature Biotechnology. The genome sequence and gene annotation can be browsed through the BOGAS system.
Methylotroph
Methylotrophs are a diverse group of microorganisms that can use reduced one-carbon compounds, such as methanol or methane, as the carbon source for their growth; and multi-carbon compounds that contain no carbon bonds, such as dimethyl ether and dimethylamine...
ic yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
. Pichia is widely used for protein expression
Protein expression
Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...
using recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
techniques. Hence it is used in biochemical
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and genetic
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
research in academia
Academia
Academia is the community of students and scholars engaged in higher education and research.-Etymology:The word comes from the akademeia in ancient Greece. Outside the city walls of Athens, the gymnasium was made famous by Plato as a center of learning...
and the biotechnical industry
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
.
Pichia pastoris as an expression system
Pichia pastoris is frequently used as an expression system for the production of proteins. A number of properties makes P. pastoris suited for this task: P. pastoris has a high growth rate and is able to grow on a simple, inexpensive medium. P. pastoris can grow in either shake flasks or a fermenter, which makes it suitable for both small and large scale production.Pichia pastoris has two alcohol oxidase
Alcohol oxidase
In enzymology, an alcohol oxidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are primary alcohol and O2, whereas its two products are aldehyde and H2O2....
genes, AOX1 and AOX2, which have a strongly inducible
Regulation of gene expression
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation.Regulation of gene expression includes the processes that cells and viruses use to regulate the way that the information in genes is turned into gene products...
promoter. These genes allow Pichia to use methanol as a carbon
Carbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
and energy source. The AOX promoters are induced by methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
and are repressed by e.g. glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
. Usually the gene for the desired protein is introduced under the control of the AOX1 promoter, which means that protein production can be induced by the addition of methanol. In a popular expression vector, the desired protein is produced as a fusion product to the secretion
Secretion
Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...
signal of the α-mating factor from Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
(baker's yeast). This causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification
Protein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...
. There are commercially available plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s that have these features incorporated (such as the pPICZα vector).
Comparison to other expression systems
In standard molecular biologyMolecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
research, the bacterium Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
is the most frequently used organism for production of recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
and proteins. This is due to E. coli's fast growth rate, good protein production rate and undemanding growth conditions. Protein expression in E. coli is usually faster than in Pichia pastoris for several reasons: Competent E. coli cells can be stored frozen, and thawed immediately before use, whereas Pichia cells have to be produced immediately before use. Expression yields in Pichia vary between different clones
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
, and usually a large number of clones needs to be screened for protein expression before a good producer is found. Optimal induction times of Pichia are usually on the order of days, whereas E. coli usually reaches optimal yields within hours of induction. The major advantage of Pichia over E. coli is that Pichia is capable of producing disulfide
Disulfide
In chemistry, a disulfide usually refers to the structural unit composed of a linked pair of sulfur atoms. Disulfide usually refer to a chemical compound that contains a disulfide bond, such as diphenyl disulfide, C6H5S-SC6H5....
bonds and glycosylation
Glycosylation
Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...
s in proteins. This means that in cases where disulfides are necessary, E. coli might produce a misfolded protein, that is usually inactive or insoluble.
The well-studied Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
(baker's yeast) is also used as an expression system with similar advantages over E. coli as Pichia. However Pichia has two main advantages over S. cerevisiae in laboratory and industrial settings:
Firstly, Pichia, as mentioned above, is a methylotroph
Methylotroph
Methylotrophs are a diverse group of microorganisms that can use reduced one-carbon compounds, such as methanol or methane, as the carbon source for their growth; and multi-carbon compounds that contain no carbon bonds, such as dimethyl ether and dimethylamine...
, meaning it can grow with the simple alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....
methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
as its only source of energy — Pichia can easily be grown in cell suspension in reasonably strong methanol solutions that would kill most other micro-organisms, a system that is cheap to set up and maintain.
Secondly, Pichia can grow to very high cell densities, and under ideal conditions can multiply to the point where the cell suspension is practically a paste. As the protein yield from expression in a microbe is roughly equal to the product of the protein produced per cell and the number of cells, this makes Pichia of great use when trying to produce large quantities of protein without expensive equipment.
Compared to other expression systems such as S2-cells from Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
or Chinese Hamster Ovary cell
Chinese Hamster Ovary cell
Chinese hamster ovary cells are a cell line derived from the ovary of the Chinese hamster. They are often used in biological and medical research and commercially in the production of therapeutic proteins. They were introduced in the 1960s and grow as a cultured monolayer...
s, Pichia usually gives much better yields. Cell lines from multicellular organisms usually require complex rich media, including amino acids, vitamin
Vitamin
A vitamin is an organic compound required as a nutrient in tiny amounts by an organism. In other words, an organic chemical compound is called a vitamin when it cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet. Thus, the term is conditional both on...
s and growth factor
Growth factor
A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes....
s. These media significantly increase the cost of producing recombinant proteins. Additionally, since Pichia can grow in media containing only one carbon source and one nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
source, it is suitable for isotopic labelling applications in e.g. protein NMR. However, a number of proteins require chaperonin
Chaperonin
Chaperonins are proteins that fold and unfold other proteins. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones...
s for proper folding. Thus, Pichia is unable to produce a number of proteins for which the host lacks the appropriate chaperones. In 2006 a research group has managed to create a strain that produces Erythropoietin
Erythropoietin
Erythropoietin, or its alternatives erythropoetin or erthropoyetin or EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production...
in its normal glycosylation form. This was achieved by exchanging the enzymes responsible for the fungal type glycosylation, with the mammalian homologs. Thus, the altered glycosylation pattern allowed the protein to be fully functional.
Pichia pastoris as a model organism
Another advantage of Pichia pastoris is its similarity to the well-studied Saccharomyces cerevisiaeSaccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
(also known as baker's yeast). As a model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
for biology, and having been used by man for various purposes throughout history, S. cerevisiae is well studied, to say the least. The two yeast species (Pichia, Saccharomyces) have similar growth conditions and tolerances, and thus the culturing of Pichia pastoris can be readily adopted by labs without specialist equipment.
The Pichia pastoris GS115 genome has been sequenced by the Flanders Institute for Biotechnology (VIB) and Ghent University (UGent) and published in Nature Biotechnology. The genome sequence and gene annotation can be browsed through the BOGAS system.