Protein expression system
1. Cell free: In vitro, using biomolecular translation machinery extracted from cells. eg-; E. coli, Rabbit Reticulocyte (RRL), Wheat germ, insect, Human. 2. Bacterial: popular, easy to culture, wide range of Host, Vector etc. produce commercially, often non functional Eucaryotic protein. 3. Eucaryotic
I. Yeast: Simplest Eucaryotes
II. Insect: Baculovirus Expression System,
Insect Select StableExpression System DES: Drosophila StableExpression System I. Mamalian: produce functional protein, low yield, high cost of production, not conducive to either high throughput protein synthesis or expression of proteins that are toxic to host cells. Genetic Elements Essential for Expression
Eukaryotic proteins produced in bacteria may encounter following problems * Unstable or lack biological activity due to lack of posttranslational modifications like glycosylation or correct assembly. * Possess unacceptable contaminants after purification, Bacterial compounds that are toxic to the humans and animals. * (Pyrogens) may contaminate the final product.
* Eukaryotic expression systems developed for production of proteins which would be identical to the natural protein in its biophysical, biochemical and functional properties and can be used as therapeutic agents in either Humans or Animals. Expression systems for eukaryotic protein
Yeast: S. cerevisiae: detect protein-protein interactions by two-hybrid assay. Pichia pastoris: high-level protein production. Baculovirus: insect tissue culture transiently infected with recombinant virus containing the gene of interest high expression levels can express and assemble large proteins efficient cleavage of signal peptides and processing of the protein post-translational modifications simultaneous expression of multiple genes. Mammalian tissue culture: Relatively low expression levels suitable for cell-based functional studies (signal transduction, apoptosis, co-IP) typically requires serum-containing medium. Advantages of protein expression in yeast
* Extremely useful for expression and analysis of Eukaryotic proteins. * Molecular-biological research into yeast is well developed. * Genetically well characterized and are known to perform many post-translational modifications. * Easy and less expensive to work with compared to insect or mammalian cells * Grow quickly in defined medium.
* Can be adapted to fermentation.
* Yeast expression systems are ideally suited for large-scale production of recombinant Eukaryotic proteins. Introduction
* Genetic engineering, i.e. transformation of yeast cells with recombinant DNA, became feasible for the first time in 1978 [Beggs, 1978; Hinnen et al., 1978].
* Generally these plasmid vectors (shuttle vectors) contain genetic material derived from the E.coli vector pBR322 (or its derivatives) and a genetic element (origin of replication) which enable them to be propagated in E.coli cells prior to transformation into yeast cells and a selectable marker (mainly the ß lactamase gene, amp) for the bacterial host . * Engineering of yeast hosts with the capability to add humanized N-glycans. Saccharomyces cerevisiae as a Expression System
* It is single celled.
* Extremely well known genetically and physiologically.
* It can be grown readily in small culture vessel and large scale bioreactors. * Several strong promoters isolated and characterized.
* It can carry out many post translational modifications. * A number of proteins that have been produced in S. cerevisiae are currently being used commercially as vaccines, pharmaceuticals and diagnostic agents. Expression in eukaryotic host requires
1. Host strain.
3. Specific origin of replication
4. Selective marker.
5. Promoter etc.
1. Yeast integrated plasmid(YIP).
2. Yeast episomal plasmids (YEps).
3. Yeast replicating plasmids (YRps).
4. Yeast centromere plasmids (YCps).
5. Yeast artificial chromosomes(YACs).
All three(2,3,4) autonomous plasmid vectors are maintained in yeast as circular DNA molecules. Yeast vector
* Three classes of S. cerevisiae expression vectors described : * Episomal or plasmid vectors not good under conditions of large scale growth * Integrating vectors stable, but copy number is limited. * Yeast artificial chromosomes designed to clone a large segment of DNA (> 100 kb) which is then maintained as a separate chromosome in the host yeast cell. The YAC system is highly stable. * A YAC vector has a sequence that acts as an origin of replication (ARS), a yeast centromere sequence and a sequence that appears at both ends after linearization of the DNA and act as chromosome telomeres to maintain chromosomal stability. Common features of the four kinds of vectors
* Contain unique target sites.
* Replicate in E. coli, often at high copy number.
* Employ markers that can be selected readily in yeast.
It is necessary to amplify the vector DNA in E. coli before transformation of the ultimate yeast recipient.
* A YIP plasmid consists of the basic E. coli vector plus a yeast selectable marker gene, but does not contain a Saccharomyces origin of replication. * YIP plasmids must integrate into a chromosome in order to be replicated at each cell division. * Integrative plasmids (YIp) which by homologous recombination are integrated into the host genome at the locus of the marker, when this is opened by restriction and linearized DNA is used for transformation. This (normally) results in the presence of one copy of the foreign DNA inserted at this particular site. * YIp plasmids with two marker YFG1 (your favourite gene also present on the yeast genome and hence a homologous site) and URA3 have the potential to integrate at either of the genomic loci. * URA 3 orotidine 5-phosphate decarboxylase (ODCase) involved in the synthesis of pyrimidine, Loss of ODCase activity leads to a lack of cell growth unless uracil or uridine is added to the media. Yeast Episomal Plasmids Yep
* A YEp plasmid contains an origin of replication derived from 2μm circle in the basic YIP vector. Constructing strategy-
* An E. coli cloning vector .
* The naturally occurring yeast 2 μm plasmid.
* Multiple copies of the transformed plasmid are propagated in the yeast cell and maintained as episomes. * Use for small scale expression studies.
Yeast replicating plasmids YRp
* Constructed by adding Saccharomyces origin of replication derived ARS to YIP vector. * Transform yeast very efficiently.
* The transformants are very unstable often left in the mother nucleus. * ARS allows the transformed plasmids to be propagated several hundred-fold. Yeast Centromere Plasmids YCp
* A YCp plasmid contains a centromere sequence, CEN, added to a YRp vector. * The plasmids are treated like mini chromosomes by the dividing yeast cell. * attach to spindle fibers and transmitted to both mother and daughter cells in mitosis and meiosis,. * These are mitotically stable in the absence of selective pressure, segregate during meiosis in a Mendelian manner, found at low copy number in the host cell. * Used as regular cloning vectors (e.g., pYC2, pBM272)
Yeast artificial chromosomes YACs
* Used to carry large chromosomal DNA fragments.
* Useful in cloning fragments for various genome sequencing projects and for positional cloning studies.
* The construction of YACs follow a similar strategy as that of the ARS/CEN plasmids. * In addition to the usual components, they are endowed with telomere sequences flanking a yeast marker gene (HIS3 in pYAC4); restriction sites flanking the telomere sequences can later be used to linearize the plasmid DNA for yeast transformation. * The insertion site for large foreign DNA segments is located within a second ‘marker’ gene, the SUP4 suppressor tRNA, which allows selection of transformed cells that possess the appropriate genetic background. * As the linearized plasmids behave like endogenous chromosomes, they are maintained and replicated in the same manner as resident yeast chromosomes. * Note : SUP4 gene encoding a UAA suppressor tRNA by incorporatine tyrosine(UAU)
* Antibiotic agents like ; kanamycine(G418) and Hygromycin, tetracycline. * G418 blockspolypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells. Resistance to G418 is conferred by the neo gene from Tn5 encoding an aminoglycoside 3′-phosphotransferase, APT 3′ II * Hygromycin is active against both prokaryotic and eukaryotic cells. It stabilizes the tRNA-ribosomal acceptor site. * Tetracycline is active against both prokaryotic and eukaryotic in procaryotes its binds to 30s subunit whereas in eucaryotes it binds to 80 Ssubunit. Selectable Marker
* Nutritional genes- The most widely used markers: URA3, LYS2, ADE1, ADE2, HIS3, LEU2, TRP1. * The URA3 and LYS2 yeast genes have a marked advantage because both positive and negative selections are possible * Positive selection is carried out by auxotrophic(wild strain) complementation of the ura3 and lys2 mutations. * Negative selection is based on specific inhibitors, 5-fluoro-orotic acid (FOA) and α-aminoadipic acid, respectively, that prevent growth of the wild strains. Negative selection
* URA3 encodes orotidine-5’phosphate decarboxylase, an enzyme which is required for the biosynthesis of uracil. Ura3- (or ura5-) cells can be selected on media containing FOA. The URA3+ cells are killed because FOA appears to be converted to the toxic compound 5-fluorouracil by the action of decarboxylase, whereas ura3- cells are resistant. * LYS2 encodes α-aminoadipate reductase, an enzyme which is required for the biosynthesis of lysine. Lys2- and lys5- mutants, but not normal strains, grow on a medium lacking the normal nitrogen source, but containing lysine and α-aminoadipate (aAA). Apparently, lys2 andlys5 mutations cause the accumulation of a toxic intermediate of lysine biosynthesis that is formed by high levels of aAA, but these mutants still can use aAA as a nitrogen source. * The ADE1 and ADE2 yeast genes encode phosphoribosylamino – imidazole-succinocarbozamide synthetase and phosphoribosylamino-imidazole-carboxylase, respectively, two enzymes in the biosynthetic pathway of adenine. ade1 and ade 2 mutant, but no other ade-mutants, produce a red pigment that is apparently derived from the polymerization of the intermediate phosphoribosylamino – imidazole (denoted AIR).
Mainly Used Vector And Marker in Yeast
Promoter of Yeast Expression Vectors
* Regulated Promoters
* Yeast expression vectors will employ promoter and terminator sequences in addition to the gene of interest. * It is advantageous to use yeast-derived (homologous) rather than heterologous sequences,because the former are more efficient, and heterologous elements will sometimes not work in yeast. * Constitutive promoters are derived from genes of the glycolytic pathway, because these lead to high-level transcriptional expression. Mostly used Promoter for Expression
Origin of replication
* 2 µm and ars1 are commonly used origin of Replication for autonomous replication of vectors outside of the chromosomes. * 2 µm ori is origin of replication in 2 µm plasmmid in s.cerevisiae and ars1 is chromosomal region (ARS)of s. Pombe. * When stb ori is combined with ars1, the resulting vector can be maintained more stably in S. pombe as multi-copy vectors more than 200 copies per cell. Secretion Signals in Yeast
* Prepro alpha factor-mating pheromone(MF1 and MF2).
* HSp150- yeast Hsp150 protein has a special ticket out of the cell. compensates for deficiencies in the coat protein and transport protein. * PHO1;-transfer of inorganic phosphate.
* SUC2;- sucrose transporters and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. * KILM1 (killer toxin type 1)- The relative secretion levels were as follows: αF . KILM1 . PHO1 and SUC2. * GGP1- encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The Yeast Two-Hybrid System
* The yeast Two-hybrid system has been developed as a potent tool to identify cDNAs, carried on one plasmid, which code for proteins that interact with a target protein specified by a DNA sequence carried on another plasmid. * The two-hybrid assay is based on the fact that the yeast Gal4p transcriptional activator is composed of two physically separable, functionally independent activation and binding domains (Gal4-AD and Gal4-BD, respectively). * The cloning vectors, which are endowed with different markers, are used to create fusions of the GAL4 domains with genes for proteins that potentially interact . Principle of the yeast two hybrid system
* After introduction into a yeast strain that carries an appropriate reporter gene (HIS3 or lacZ) with a GAL4 UAS element in its promoter, if the two domains interact, the DNA-BD will be tethered to the AD, and will reconstitute the Gal4 transcriptional activator, which results in the activation of the reporter gene. * Selection can be made by screening for His+ or lacZ+ positives, and the GAL4-AD/library fusion plasmid can efficiently be retrieved from such colonies. * The method has been improved since its invention, particularly to minimize the appearance of false positives, which however still seems to be a problem not completely overcome. Heterologous Protein Secretion by S. cerevisiae
* In yeast only secreted proteins are glycosylated so a secretion system must be used for heterologous proteins that require O-linked or N-linked sugars for biological activity. * Gene must encode leader to pass through secretory system.T he leader peptide is removed by a yeast endoprotease that recognizes the dipeptide Lys-Arg. Also aids in correct disulfide bond formation, proteolytic cleavage of leader, etc. occur * Over expression of PDI secretory enzyme also helps (up to 10X).
Why Other Yeast Species?
* S. cerevisiae sometimes hyperglycosylates protein.
* Proteins also sometimes retained in periplasmic space
* S. cerevisiae also produces ethanol at high cell densities which is Toxic to cells * Instabilities of recombinant strains
* For correct folding, formation of disulphide linkage and glycosylation. * Secretion of the proteins (human antithrombin III, gastric lipase, placental alkaline phosphatase and S. Cerevisiae invertase).
Pichia pastoris as host
* The methanotropic yeat P. pastoris can be grown easily and economically in large bioreactors. * Highly efficient promoters available
* Tight control (e.g. AOX1 promoter) (alcohol oxidase) * Produce up to 30% of total cell protein by wt.
* AOX1 (alcohol oxidase for methanol metabolism) promoter easily turned on by methanol. In the absence of methanol, no alcohol oxidase is synthesized. * Does not produce ethanol at high cell density.
* Secretes few proteins, simplifying purification
* Non-fermentation yeast species
* Can be grown to very high cell densities and 10- to 100-fold high-level protein expression has been achieved. * Its differ from Saccharomyces, thet this is similar to mammalian cell transformation where non homologus recombination is strongly favourred. * strong AUG1(alcohol utilization gene) promoter (now designated MOD1) from methanolica is induced by alcohol whereas is tightly repressed in medium containing glucose or glycerol * When cells are shifted to medium with methanol as the sole carbon source, transcription is rapidly induced to very high levels. Pichia methanolica
* As a result, the P. methanolica Expression System is well suited to high-level production of recombinant proteins that are toxic to the cell * The P. methanolica expression vectors, pMET and pMET , offer the following features: 1. Strong AUG1 promoter for high-level, tightly-regulated expression 2. The α-factor signal sequence for secretion of the expressed protein (in pMET only) 3. C-terminal V5 epitope for convenient detection with an Anti-V5 Antibody 4. C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin Fission yeast Schizosaccharomyces pombe as a host
* S. pombe has many similar characteristics to higher eukaryotic cells. * Some Promoters derived from mammalian cells work effectively in S. pombe. * Most intensely studied and wellcharacterized yeast species in terms of molecular genetics and cell biology. * RNA splicing mechanism, Transcription-initiation mechanism, signal-transduction system shows similarity to higher eucaryotes. Hansanuela polymorpha.
* Cultured in large scale fermenters to high cell densities * withstand temperature upto 49 o c and pH- 2,5 – 6,5 * Hansenula efficiently secrets proteins with a molecular weight of up to 150 kDa. * highest productivity values ever seen for a yeast derived protein – 13,5 g/L food supplement phytase. * Pharmacautical proteins;-Insulin, hirudin, hepatitis B vaccines or IFN α-2a for the treatment of hepatitis C etc. * use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway Hansanuela polymorpha
* Synthetic minimal medium (SYN6) has been developed for fermentation. * H. polymorpha the FMD (formate dehydrogenase) and MOX , methanol utilization promoter is used. * H. polymorpha strains expressing a GFP reporter gene under control of the FMD promoter were screened applying glucose- or glycerol-supplemented media to strain culturing. Arxula adeninivorans (Blastobotrys adeninivorans)
* No industrial processes have been developed so far.
* Only a limited range of strong promoter elements exists. * Suitable for heterologous gene expression.
* Can utilize adenine, xanthine, uric acid, putrescine and nalkylamines as carbon, nitrogen or energy sources in addition to glucose. Arxula adeninivorans
* Temperature-dependent dimorphism with mycelial structures formed at temperatures above 42°C. * Under control of the constitutive A. adeninivorans-derived TEF1 promoter * Most of the current expression studies are based on wild type strain LS3 or its leucine-auxotrophic derivative G1211 Candida boidinii
* Expression host for secretory enzyme production.
* Alcohol oxidase (AODl) promoter is used.
* Yarrowia lipolytica is a dimorphic yeast that can grow on a wide range of substrates. It has a high potential for industrial applications but there are no recombinant products commercially available yet. Kluyveromyces lactis
* Kluyveromyces lactis is a yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others to the production of chymosin (an enzyme that is usually present in the stomach of calves) for the production of cheese. Production takes place in fermenters on a 40,000 L scale. *
Yeast expression system can be applied to the following applications or research * Antigens and Ligands: for immunization and affinity purification of antibodies developed * Protein Standards: ELISA calibrators, mass spectrometry references, activity measures, and functional controls * Binding Assays: Yeast-2-hybrid assays, nuclear magnetic resonance imaging, protein-protein interactions, high-throughput screening, dye-binding assays, neutralization, and affinity determination; * Cell Based Assays: Proliferation assays, apoptosis assays, and hybridoma screening * Clinical Products: Therapeutics and vaccines.