Expression vectors

Expression of recombinant proteins

1. Expression Vectors Dr Ravi Kant Agrawal , MVSc , PhD Senior Scientist (Veterinary Microbiology) Food Microbiology Laboratory Division of Livestock Products Technology ICAR-Indian Veterinary Research Institute Izatnagar 243122 (UP) India

2. Expression Vectors: Vectors that can yield the protein products of the cloned genes. Two elements that are required for active gene expression: a strong promoter and a ribosome binding site near an initiating ATG codon . The main function of an expression vector is to yield the product of a gene, therefore a strong promoter is necessary. The more mRNA is produced, the more protein product is made.

3. Expression in E. coli E. coli  is one of the most widely used expression host for protein expression. The techniques for expression 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.

4. Expression in E. coli : Advantages: Rapid doubling time (approximately 30 min) Growth in simple defined (and inexpensive) media, An extensive knowledge of its promoter and terminator sequences Proteins of both prokaryotic and eukaryotic origin can be produced within the organism. Many proteins of prokaryotic are originated in E. coli due to E.coli cells are easily broken for the harvesting of proteins produced within the cell ( Easy harvesting). Drawback: E. coli cells are Unable to process introns and 2. Do not possess the extensive post-translational machinery ( glycosylation , methylation , phosphorylation etc).

5. In expression vector various Genetic elements required are: Origin of replication Selective marker Transcriptional promoter Unique multiple cloning sites Translational initiation regions (TIRs) Translational terminator

6. Expression vector

7. Origin of replication: Sequences that allow their autonomous replication within the cell. There are 2 types: 1) ColE1 replicon : - pBR322 (copy no. ~15-20) - pUC (copy no. ~500-700) 2) p51A replicon : -pACYC184 (copy no. ~10-12)

8. Selective marker: sequences encoding a selectable marker that assures maintenance of the vector in the host. Ampicillin Kanamycin interfere with Chloramphenical protein synthesis Tetracycline

9. Transcriptional promoter: Promoter is a – Region of DNA that control the transcription of a particular gene. It locate upstream of the genes. Recognizing site for RNA polymerase (sigma subunit). Good promoter should be: Strong promoter: Accumulation of expressed protein up 10 to 30% or more of the total cellular protein. Minimal basal expression level: Tight regulation of the promoter Easily induction: Simple and cost effective manner

10. Multiple cloning site: It is defined as a short segment of DNA which contain many restriction sites (usually 20+). To simplify the insertion of the heterologous gene in the correct orientation within the vector. Translational initiation regions (TIRs): Located on the 5’-mRNA Translational terminator: Located on the 3’-mRNA Use of Rho-independent termination

11. Some commonly used expression host-vector system and expression inducible expression system:

12. Many different promoter sequences have been used to illicit inducible protein production in E. coli 1. lac Promoter 2. tac Promoter 3. λ PL Promoter 4.T7 Expression System

13. Many different promoter sequences have been used to induce protein production in E.coli. These are: The lac Promoter: Lac prompter provides a mechanism for inducible gene expression. Without lactose in the cell, the repressor protein binds to the operator and prevents the read of RNA polymerase into the three structural genes. With lactose in the cell, lactose binds to the repressor. This causes a structural change in the repressor and it loses its affinity for the operator. Thus RNA polymerase can bind to the promoter and transcribe the structural genes. In this system lactose acts as an effector molecule. Fusing the lac promoter sequence to another gene will result in the lactose-(or IPTG-) dependent expression of that gene.

14. Control Circuit for the  lac  Operon: I P O || Z | Y | A | controlling region ǁ structural genes lac  Operon Gene Gene function I Gene for repressor protein P Promoter O Operator lac Z Gene for ß- galactosidase lac Y Gene for ß- galactoside permease lac A Gene for ß- galactoside transacetylase Operon - a cluster of structural genes that are expressed as a group and their associated promoter and operator

15.  The  lac   operon  promoter (which regulates the transcription of  lacZ , not  lacI ) (a) Presented so that the genome sequence numbers increase from left to right.  Its 5' to 3' sequence is the same as in the database (ACCESSION  AE000141 ).  (b) Presented so that the promoter sequence numbers increase from left to right.  Its 5' to 3' sequence is comparable to the  consensus sequence . 

16. lac promoter- lactose- (or IPTG- isopropylthiogalactoside ) inducible Problems with lac promoter- Weak 2. Leaky expression mutant versions of the lacI gene : lacIq allele increased DNA binding overproduction of LacI reduced level of transcription in the absence of inducer

17. The tac Promoter: The lac promoter is weak because the -35 region deviates from the consensus. The creation of a fusion sequence containing the -35 region of the E. coli trp operon and the -10 region of the lac operon controlling the expression of the genes responsible for tryptophan biosynthesis and lactose metabolism which results in the formation of the tac promoter. The tac promoter is 5 times stronger then lac promoter. Expression vectors that carry the tac promoter also carry the lacO operator and usually the lacI gene encoding the Lac repressor. Because of this, these vectors are IPTG inducible and can be repressed and induced in a variety of E. coli strains.

18. -35 CONSENSUS -10 CONSENSUS 5’-TTGACA-3’ 5’-TATAAT-3’ Lac : 5’-…TTTACAC…..TCCG GCTCGTATATTGTGT…………..CAGGAAACAGC T ATG…-3’ _______ ______ _____ ______ -35 -10 RBS Protein Trp : 5’-… TTGACAATTAATCATCG AAC….TTAACTAG………….AAAGGGTAT…….ACA ATG…-3’ _______ _______ _____ ______ -35 -10 RBS Protein Tac : 5’-… TTGACAATTAATCATCG GCTCGTATATTGTGT………AGGAAACAGCG G ATG…-3’ _______ ______ _____ ______ -35 -10 RBS Protein DNA sequence of lac, trp and tac promoter. The consensus E. coli -35 and -10 sequence based on the analysis of naturally occurring promoters are shown and the sequence of each of the promoters, extending from the -35 region to translational start site, are shown. The tac promoter is a hybrid of the trp and lac promoter. The -35 and -10 region contains closely resemble the consensus sequences. The tac promoter is able to induce the expression of target genes such that encoded polypeptide can accumulate at the level of 20-30% of the total cell protein.

19. 3. The λ Pl Promoter: This is responsible for transcription of left-hand side of the λ genome, including N and cIII . Λ repressor which is the product of the cI gene, repress the promoter. Two basic methods are used to activate this promoter. Transform the expression vector into an E. coli strain in which the cI gene has been placed under the control of tightly regulated trp promoter. Then expression of the target gene can be induced by the addition of tryptophan to the growth media, which will prevent transcription of the cI gene and consequently activate the strong λ Pl promoter. This system can be used to express highly toxic proteins. The λPL promoter - transcription of the left-hand side of the λ genome, including N and cIII

20. One way: expression vector Transform In E. coli strain ( cI gene under control of trp promoter ) Expression of the target gene - By addition of tryptophan Advantage- Tightly controlled system so can be use for highly toxic proteins cI gene - λ repressor, The λ PL Promoter Activation of the λPL promoter .

21. 1. Temperature-sensitive mutant of cI (Ci857) above 30 ◦C mutan λ repressor λPL for the expression -target genes . Advantage- high levels of target gene expression, Disadvantage- Heat pulse difficult to control Other way:

22. 4. The T7 Expression System- It is different from its E. coli counterpart. E. coli enzyme- Has a α2β2 subunit structure T7 RNA polymerase- Binds to DNA 17 bp promoter sequences (5-TAATACGACTCACTATA-3) found upstream of the T7 viral gene it activates. bacteriophage T7 RNA polymerase single-subunit

23. T7 promoter . The target gene plasmid wild-type no expression E. coli strain lac promoter T7 RNA polymerase T7 gene 1 expression of the target gene. To control the leaky production of T7 RNA polymerase (thereby ensuring that target gene expression is minimized), E. coli cells co-transformed With The plasmid pLysS - T7 lysozyme , natural inhibitor of T7 RNA polymerase.

24. The T7 Expression System: pET vector system The pET System is the most powerful system yet developed for the expression of recombinant proteins in E. coli . Translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression. The desired product can comprise more than 50% of the total cell protein a few hours after induction. pET vector: T7 promoter for expression lac O, lac I : tighter regulation of transcription origin of replication : pBR322 selectable marker : Amp r ccd B : control of cell death RBS : ribosome binding site Start codon : ATG Tag, Fusion : 6xHis , V5 Epitope, GST T7 terminator

25. The expression vector ( pET ) contain the target gene under the control of T7 (RNA polymerase) promoter. The vector is transformed into an E. coli strain that contains a copy of gene for T7 RNA polymerase gene under the control of the lac promoter (BL21 DE3). The promoter for both the target gene and T7 RNA polymerase gene, also contain the lacO operator sequence and therefore inhibited by the lac repressor( lacI ). IPTG induction allows the transcription of the T7RNA polymerase gene whose protein product activates the expression of target gene. After induction sufficient T7 RNA polymerase is produced.

26. Expression in Yeast Advantages- 1. Easy to manipulate as E. coli 2. Yeast cell growth is faster, easier and less expensive than other eukaryotic cells 3. Post-translational modifications 4. Gives higher expression levels . Three main species of yeast are used for the production of recombinant proteins – Saccharomyces cerevisiae Pichia pastoris and Schizosaccharomyces pombe

27. Baker’s yeast, S. cerevisiae , is- A single-celled eukaryote that grows rapidly (a doubling time of approximately 90 min) in simple, defined media similar to those used for E. coli cell growth. Advantage: Many, but not all, of the post-translation modifications found in higher eukaryotic cells. A number of constitutive promoters for the genes encoding phosphoglycerate kinase ( PGK), glyceraldehyde-3-phosphate dehydrogenase ( GPD) and alcohol dehydrogenase (ADH1) have been used to produce target proteins, however, these suffer with similar problems as constitutive E. coli expression. 02 Systems have been utilized for inducible expression. Saccharomyces cerevisiae

28. The GAL system – In yeast, galactose is converted to glucose -6- phosphate by the enzymes of the Leloir pathway. Each of the Leloir pathway structural genes (collectively called Gal genes) are expressed at a high levels, representing 0.5-1% of the total cellular RNA but ONLY when the cells are grown on galactose as the sole carbon source. Each of the gal genes contains within its promotor at least one or multiple binding sites for the transcriptional activator Gal4p. The binding of Gal4p and its transcriptional activity when bound is regulated by the source of carbon available. When yeast is grown on glucose, transcription of Gal4p is down regulated, so there is less Gal4p and reduced level of activator binding at the promotors of the Gal structural genes. To produce a target protein in S. cerevisiae using galactose induction, the gene encoding the protein must be cloned so that it is under the control of a Gal promotor . The promotor for Gal1 gene encoding galactokinase is most commonly used. Synthestic promotors containing multiple Gal4p binding sites are also available.

29. Expression vector is transformed in to yeast cells and protein production is initiated by switching the cells to a galactose containing medium. Protein produced by these methods is usually in low abundance and maximum production may represent 1-5% of the total cell protein. Therefore detection by CBB R 250 ot possible and require western blotting Additional difficulty is brought about by the activator of the GAL genes i.e. Gal4p which is normally present in the yeast cells at very low level. Therefore if expression vector which carries multiple Gal4p binding sites, as a high copy number plasmid, there may not be sufficient Gal4p to activate the expression of all the available target genes to a maximum level. To overcome this yeast strains have been constructed in which the coding sequence of the Gal4 gene has been placed under the GAL promotor control. This results in feedback loop in which induction by galactose results in the production of Gal4p so that more of the target gene may be expressed.

30. GAL genes Promoter one / more binding site for transcriptional activator Gal4p Binding of Gal4p to these sites and its transcriptional activity when bound, is regulated by the source of carbon galactose ( galactose in medium) Structural genes ( GAL genes) 0.5-1 per cent of the total cellular mRNA Leloir pathway enzymes glucose-6-phosphate

31. Less GAL structural genes yeast Glucose (preferred carbon source) less Gal4p raffinose , production Gal4p Gal80p, inhibits

32. Galactose inducible gene expression in yeast. The expression of genes from multicopy vectors under the control of the GAL1 promoter (PGAL1 ) can be increased substantially if the gene encoding the transcriptional activator of GAL1, GAL4, is also placed under the control of PGAL1. In this case, induction by galactose will produce more Gal4p and consequently more of the target protein

33. 2.The CUP1 System: Copper ions (Cu 2+ and Cu 3+ ) are essential at appropriate levels, yet toxic at higher levels in all living MOs. Cells must maintain a proper level of copper ions Copper homeostasis in S. cerevisiae involves Distribution Uptake, Detoxification mechanisms. At High concentrations, copper ion detoxification is mediated by a copper ion sensing metelloregulatory transcription factor called Ace1p. Upon interaction with copper, Ace1p binds DNA upstream of the CUP1 gene, which encodes a metallothionin protein and induces its transcription. The transcription of CUP1 is induced rapidly by addition of exogenous copper to the medium. Expression vectors harbouring CUP1 promotor can therefore be used to induce target gene expression in copper dependent fashion.

34. Cu ions Ace1p CUP1 gene metallothionein Advantage: Can be grown on rich carbon sources , such as glucose, to high cell density, and protein production is initiated by the addition of copper sulphate ( 0.5 mM final concentration) to the cultures. Drawback- Due to presence of copper ions in yeast growth media, and indeed in water supplies. Therefore off state in the absence of added copper may still yield significant levels of protein production

35. Pichia pastoris Pichia pastoris is a methylotropic yeast capable of metabolizing methanol as its sole carbon source. First step is oxidation of methanol to formaldehyde using molecular o2 utilizing alcohol oxidase (AOX1). Alcohol oxidase has poor affinity for O2 and Pichia pastoris compensates it by producing large amounts of Alcohol oxidase . Therefore, the promotor regulating the production of Alcohol oxidase may be utilized to drive heterologous protein expression in Pichia pastoris since it is tightly regulated and induced by methanol to very high levels. P. pastoric cells containing expression vector, which is usually integrated into the genome as single or multiple copies are grown in gycerol (growth on glucose represses AOX1 transcription, even in the presence of methanol), to extremely high cell density prior to addition of methanol. Once induced, target protein may accumulate at very high levels, often ranging from 0.5-10’s of grams per litre of yeast culture e.g. HBsAg is produced 1g/ litre .

36. Methylotrophic yeast methanol formaldehyde O2 alcohol oxidase ( AOX1 gene) Advantages- Tightly regulated High levels protein production P. pastoris has advantage of glycosylation of secreted proteins Glycoproteins produced in P. pastoris resmble closely the glycoprotein structure of those found in higher eukaryotes.

37. Schizosaccharomyces pombe : Single cell eukaryotic organism The properties like codon usage, chromosomal structure and function, cell cycle control and RNA spilcing suggest that S. pombe is an ideal candidate for production of eukaryotic proteins. Additionally, eukaryotic proteins produced in S. pombe are more likely to be folded properly which reduce protein insolubility. Protein production in S. pombe is usually controlled by nmt1 promotor . This promotor is active when cells are grown in absence of thiamine. In the presence of more than 0.5 uM thiamine, promotor is turned off. Overall protein production levels- similar to S. cerevisiae . Advantages- expressed proteins are folded properly Solubility more No message in thiamine (nmt1) promoter thiamine > 0.5 μM thiamine, the promoter is turned off.

38. Using animal cells for recombinant protein production The difficulties inherent in synthesis of a fully active animal protein in a microbial host have prompted biotechnologists to explore the possibility of using animal cells for recombinant protein synthesis. For proteins with complex and essential glycosylation structures, an animal cell might be the only type of host within which the active protein can be synthesized.

39. Insect Cell Expression System Higher Eukaryotic system than yeast - more complex post-translational modifications . Folding of mammalian proteins - soluble protein mammalian origin. The most commonly used vector system - baculovirus . The benefits of protein expression with baculovirus are: Eukaryotic post-translational modification Proper protein folding and function High expression levels

40. Expression in Insect Cells The expression system is based on the baculoviruses , a group of viruses that are common in insects (& insect cell lines) but do not normally infect vertebrates. The baculovirus genome includes the polyhedrin gene, whose product accumulates in the insect cell as large nuclear inclusion bodies toward the end of the infection cycle . The product of this single gene frequently makes up over 50% of the total cell protein. Similar levels of protein production also occur if the normal gene is replaced by a foreign one. Baculoviruses - rod-shaped viruses. Nuclear polyhedrosis viruses (NPV) - occlusion bodies ( polyhedrin ). The polyhedrin gene is transcribed at very high levels late in the infection process (3-5 d post-infection). The polyhedrin promoter can be used to drive target gene expression.

41. Baculoviruses are rod-shaped viruses that infect insects and insect cell lines. They have double-stranded circular DNA genomes in the range of 90–180 kbp (Ayres et al., 1994). Viral infection results in cell lysis , usually 3–5 d after the initial infection, and the subsequent death of the infected insect. The NUCLEAR POLYHEDROSIS VIRUSES are a class of baculoviruses that produce occlusion bodies in the nucleus of infect cells. These occlusion bodies consist primarily of a single protein, polyhedrin , which surrounds the viral particles and protects them from harsh environments. Crystalline inclusion bodies in the nuclei of insect cells infected with a baculovirus.

42. Most viruses of this type need to be eaten by the insect before infection will occur, and the occlusion body protects the viral particles from degradation in the insect gut. The polyhedrin gene is transcribed at very high levels late in the infection process (2–4 d post-infection). In cultured insect cells, the production of inclusion bodies is not essential for viral infection or replication. Consequently, the polyhedrin promoter can be used to drive target gene expression. The baculovirus Autographa californica nuclear polyhedrosis virus ( AcNPV ) has become a popular tool of the production recombinant proteins in insect cells (Fraser, 1992). It is used in conjunction with insect cell lines derived from the moth Spodoptera frugiperda . These cell lines (e.g. Sf9 and Sf21) are readily cultured in the laboratory.

43. The size of the baculoviral genome generally precludes the cloning of target genes directly onto it. Instead, the target gene is cloned downstream of the polyhedrin promoter in a transfer plasmid (Lopez-Ferber, Sisk and Possee , 1995). The transfer plasmid also contains the sequences of baculovirus genomic DNA that flank the polyhedrin gene, both upstream and downstream. To produce recombinant viruses, the recombinant transfer plasmid is co- transfected with linearized baculovirus vector DNA into insect cells. The flanking regions of the transfer plasmid participate in homologous recombination with the viral DNA sequences and introduce the target gene into the baculovirus genome. The recombination process also results in the repair of the circular viral DNA and allows viral replication to proceed through the re-formation of ORF1629 (a viral capsid associated protein that is essential for the production of viral particles). Recombinant viral infection can be observed microscopically by viewing viral plaques on a lawn of insect cells.

44. Plaques containing recombinant virus will be unable to form occlusion bodies due to the lack of a functional polyhedrin protein (Smith, Summers and Fraser, 1983). Screening plaques this way is, however, technically difficult. Therefore, the transfer plasmids also usually contain the lacZ gene , or another readily observable reporter gene, which allows for the visual identification of recombinant plaques by their blue appearance after staining with X-Gal. Following transfection and plaque purification to remove any contaminating parental virus, a high- titre virus stock is prepared, and used to infect large-scale insect cell culture for protein production. The infected cells undergo a burst of target protein production, after which the cells die and may lyse . Protein production in baculovirus infected insect cells has the advantage that very high levels of protein can be produced relative to other eukaryotic expression systems, and that the glycosylation pattern obtained is similar, but not identical, to that found in higher eukaryotes ( Possee , 1997; Joshi et al., 2000). Baculoviruses also have the advantage that multiple genes can be expressed from a single virus. This allows the production of protein complexes whose individual components may not be stable when expressed on their own (Roy et al., 1997 ).

45. Baculovirus is a very large DNA virus (genome of about 150 kb) that infects insect cells. To express a foreign gene in baculovirus, the gene of interest is cloned in place of the viral coat-protein gene in a plasmid carrying a small part of the viral genome. The recombinant plasmid is cotransfected into insect cells with wild-type baculovirus DNA. At a low frequency, the plasmid and viral DNAs recombine through homologous sequences, resulting in the insertion of the foreign gene into the viral genome. Virus plaques develop, and the plaques containing recombinant virus look different because they lack the coat protein. The plaques with recombinant virus are picked and expanded. This virus stock is then used to infect a fresh culture of insect cells, resulting in high expression of the foreign protein. Baculovirus Expression System

46. The production of a recombinant baculoviral genome for the production of proteins in insect cells. The target gene is cloned under the control of the polyhedrin promoter into a transfer vector that also contains regions of the viral genome that flank the polyhedrin locus. The vector is then co- transfected into insect cells with a viral genome that has been linearized using restriction enzymes (RE) that cut in several places. Homologous recombination between the linear genome and the vector will result in formation of a functional viral genome that is capable of producing viral particles. The inclusion of lacZ in the transfer vector allows for visual screening of viral plaques to identify recombinants

47. The main disadvantages of producing proteins in this way is that the construction and purification of recombinant baculovirus vectors for the expression of target genes in insect cells can take as long as 4–6 weeks, and that the cells grow slowly (increasing the risk of contamination) in expensive media. An alternative approach to recombinant viral genome production uses site-specific transposition in E. coli rather than homologous recombination in insect cells ( Luckow et al., 1993 ). E. coli Baculovirus Shuttle Vector - Bacmids

48. It is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector ( bacmid ) propagated in E. coli. The bacmid contains the entire baculovirus genome, a low-copy number E. coli F-plasmid origin of replication and the attachment site for the bacterial transposon Tn7. The bacmid propagates in E. coli as a large plasmid. Recombinant bacmids are constructed by transposing a Tn7 element from a donor plasmid, which contains the target gene to be expressed, to the attachment site on the bacmid – a helper plasmid encoding the transposase is required for this function. The recombinant bacmid can be isolated from E. coli and transfected directly into insect cells.

49. E. coli Baculovirus Shuttle Vector - Bacmids Shuttle vectors allow ease of transfer between systems Genetic manipulations in one system, expression in another

50. Advantages The polyhedrin gene is not required for the continuous production of infectious virus in insect cell culture. Its sequence is replaced with that of the heterologous gene. The polyhedrin gene promoter is very strong. This determines a very high level of production of recombinant protein. Very late expression allows for the production of very toxic proteins. This system is capable of post-translational modifications.

51. Disadvantages Expensive. Glycosylation in insect cells is different (insect cells unable to produce complex N -linked side chains with penultimate galactose and terminal sialic acid) from that in vertebrate cells, therefore, a problem for therapeutic proteins. A large fraction of the RP can be poorly processed and accumulates as aggregates. Discontinuous expression: baculovirus infection of insect cells kills the host and hence the need to re-infect fresh cultures for each round of protein synthesis. Inefficient for production on a commercial scale

52. Modifying the Insect Cell Host Genetic engineering of the host for proper expression Add missing glycosylation enzymes Add proteolytic processing enzymes

53. Expression in Higher-Eukaryotic Cells For the production of mammalian proteins, mammalian cells have an obvious advantage. Mammalian cell lines derived from humans or hamsters have been used in synthesis of several recombinant proteins, and in most cases these proteins have been processed correctly and are indistinguishable from the non-recombinant versions. Two modes of expression - transient and stable. Three cell types are dominant in transient expression: human embryonic kidney (HEK), COS and baby hamster kidney (BHK), whilst CHO (Chinese hamster ovary) cells are used predominantly for stable expression. Note: The acronym "COS" is derived from the cells being  C V-1 (simian) in  O rigin, and carrying the  S V40 genetic material. [2]  Two forms of COS cell lines commonly used are COS-1 and COS-7.

54. Mammalian expression vectors Eukaryotic origin of replication is from an animal virus: e.g. Simian virus 40 (SV40). Popular markers for selection are the bacterial gene Neo r (encodes neomycin phosphotransferase ), which confers resistance to G418 ( Geneticin ), and the gene, encoding dihydropholate reductase ( DHFR ). When DHFR is used, the recipient cells must have a defective DHFR gene, which makes them unable to grow in the presence of methotrexate (MTX), unlike transfected cells with a functional DHFR gene. Promoter sequences that drive expression of both marker and cloned heterologous gene, and the transcription termination ( polyadenyation signals) are usually from animal viruses (human CMV, SV40, herpes simplex virus) or mammalian genes (bovine growth hormone, thymidine kinase ).

55. Mammalian Expression Vector “I” is an intron that enhances expression Other signals similar to insect and prokaryotic vectors

56. Translation Control Elements K - Kozak Sequence (equivalent to RBS) S - For secretion signal peptide T - tag peptide for purification P - Proteolytic cleavage sequence SC - Stop codon for translation 3’UTR - proper sequences for efficient translation and mRNA stability (e.g. polyadenylation sequence)

57. Two Vector Expression System Useful for proteins of two different polypeptides

58. Two Gene Expression Vector

59. Selectable Markers for Mammalian Systems Most commonly used to select for transformed cells (killing non-resistant ones) Can be used for increasing expression of heterologous proteins

60. Selective marker gene systems for mammalian cells

61. Use of Selectable Markers for Increasing Heterologous Protein Production in Mammalian Systems Methotrexate (MTX ) inhibits dihydrofolate reductase (DHFR) DHFR - ve host cell with DHFR gene on cloning vector (i.e. linked to target gene) Gradually increase MTX concentration in culture Gene copy number of DHFR and linked target gene increase to compensate for inhibition of DHFR (more protein that is less active gives cell enough metabolic through put to survive)

62. However, this is the most expensive approach to recombinant protein production, especially as the possible co-purification of viruses with the protein means that rigorous quality control procedures must be employed to ensure that the product is safe. The major problem with expressing genes in mammalian cells is that expression levels like those we have discussed above are simply not currently available. For many years protein production in mammalian cells has utilized strong constitutive promoters to elicit transcription of target genes. Promoters, such as those derived from the SV40 early promoter, the Rous sarcoma virus (RSV) long terminal repeat promoter and the cytomegalovirus (CMV) immediate early promoter, will all constitutively drive the expression of genes placed under their control . Inducible systems can also be used e.g. heat-shock promoters or glucocorticoid hormone inducible systems have been used to express target genes ( Wurm , Gwinn and Kingston, 1986; Hirt et al., 1992). These systems, however suffer from leaky gene expression in the absence of induction and potentially damaging induction conditions. To overcome some of the problems of using endogenous promoters to drive target gene expression, systems have been imported from bacteria to control gene expression in mammalian cells.

63. Tet -on/ Tet -off System The control of transcriptional initiation is fundamentally different between eukaryotes and prokaryotes. An activator from prokaryotes is unable to bring about a transcriptional response in eukaryotes and vice versa. DNA binding is, however, species independent . The tightly regulated DNA binding properties of prokaryotic activators can be used to direct eukaryotic activation domains to drive the expression of target genes. One such system exploits the DNA properties of the E. coli tetracycline repressor . The E. coli tet operon was originally identified as a transposon (Tn10) that confers resistance to the antibiotic tetracycline (Foster et al., 1981). The TetR protein, in a similar fashion to the lac repressor protein ( LacI ) , binds to the operator of the tetracycline-resistance operon and prevents RNA polymerase from initiating transcription.

64. Activation of the tetracycline-resistance operon occurs when tetracycline itself binds to the repressor and induces a conformational change that inhibits its DNA binding activity. The TetR protein has a very high affinity for the antibiotic (association constant ∼3 × 10−9 M−1) and will dissociate from its DNA binding site when tetracycline is present at low concentrations (Takahashi, Degenkolb and Hillen , 1991). The regulated DNA binding activity of TetR cannot itself elicit a transcriptional response in eukaryotes, but can if the protein is fused to a eukaryotic transcriptional activator domain. The use of the tet system to drive target gene expression in eukaryotes relies on the insertion of two recombinant DNA molecules into the host cell.

65. Regulator plasmid – produces a version of the E. coli tetracycline repressor ( TetR ) that is fused to the transcriptional activation domain of the herpes simplex virus VP16 protein. The fusion protein is constitutively produced in the host cell from the CMV promoter. Response plasmid – contains the target gene cloned downstream of multimerised copies of the tetracycline operator ( tetO ) DNA sequence that form a tetracycline response element (TRE) cloned into a minimal CMV promoter that is not, on its own, able to support gene activation.

66. Figure: Tetracycline regulated gene expression for protein production in mammalian cells. The Tet -off and Tet -on systems differ in their transcriptional response to added tetracycline. The Tet -off system turns transcription of the target gene off in response to tetracycline, whereas the Tet -on system, which contains a mutant version of TetR with altered DNA binding properties, activates gene expression in response to tetracycline addition

67. In the absence of tetracycline, the TetR-VP16 fusion protein will bind to the TRE and activate transcription of the target gene. Upon the addition of tetracycline to the cells, however, TetR will dissociate and target gene transcription will be turned off ( Gossen and Bujard , 1992). That is, the addition of tetracycline turns target gene expression off. The use of the tet system has become more prevalent due to the existence of a mutant version of TetR .

68. The mutant tetracycline repressor contains four amino acid changes (E71K, D95N, L101S and G102D) from the wild-type protein that radically alter its DNA binding properties. Rather than tetracycline inhibiting its DNA binding properties, the mutant protein, called rTetR for reverse tetracycline repressor, will only bind DNA in the presence of tetracycline ( Gossen et al., 1995). This means that, with the appropriate TetR fusion to the activation domain of VP16, target gene expression can either be inhibited or activated in the presence of tetracycline.

69. Tet -off uses the wild-type TetR protein fused to VP16. Target gene expression is active in the absence of tetracycline but not in its presence. Tet -on uses the mutant rTetR proteins fused to VP16. Target gene expression is active in the presence of tetracycline but not in its absence.

70. The advantage of this on and off switching system is that host cells do not need to be exposed for long times to the antibiotic prior to the induction of either gene expression or gene silencing. Additionally, the control over target gene activation achieved using the Tet system is very tight. For example, transgenic mice have been produced that carry the diptheria toxin A gene under the control for a TRE promoter. Small quantities of the toxin, perhaps as little as a single molecule, will lead to cell death. When fed with water containing tetracycline, mice containing a Tet -off version of the regulator in conjunction with the diptheria toxin responder are healthy until tetracycline is removed, when death ensues as a result of toxin production (Lee et al., 1998).

71. Advantages: There are no examples of higher eukaryotic proteins, which could not be made in detectable levels, and in a form identical to the natural host (that includes all types of post-translational modifications).  Disadvantages: Cultures characterized by lower cell densities and lower growth rates. Maintenance and growing very expensive. Gene manipulations are very difficult. Mammalian cells might contain oncogenes or viral DNA, so recombinant protein products must be tested more extensively

72. Pharming - recombinant protein from live animals and plants The use of silkworms for recombinant protein production is an example of the process Often referred to as pharming, where a transgenic organism acts as the host for protein synthesis. Pharming is a recent and controversial innovation in gene cloning.

73. Applications Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools.  Functional analysis of cloned genes. Gene cloning in yeast systems. Protein expression trials. Protein purification.

74. Recombinant Proteins Successfully Produced in S. cerevisiae For a range of reasons as expressed previously each of these represented a better product than was obtainable using a prokaryotic expression system

75. Examples of Proteins Successfully Produced by Baculovirus Systems

76. Thanks Acknowledgement: All the material/presentations available online on the subject are duly acknowledged. Disclaimer: The author bear no responsibility with regard to the source and authenticity of the content. Questions???