Manipulation of gene Expression in Bacteria EXPRESSION EXPRESSION EXPRESSION Biotechnology The primary objective of gene cloning or genetic engineering is to express the cloned gene in a selected host organism Host System Bacteria Yeast Insect Mammalian cells GENETICALLY ENGINEERED PRODUCTS Some products produced by genetic engineering and are currently on the market – Bt potatoes – PRV Papaya – Herbicide resistant corn – Human growth hormone – Insulin – human interferon protein – monoclonal antibodies Gene expression in different systems Bacterial genes being expressed in a eukaryotic system Eukaryotic genes are being expressed in bacteria Expression vectors Vectors designed to overproduce specific gene products Use well-characterised RNA and protein synthesis systems – Lambda ZAPII – pTrc99A – pBluescript II SK+ Manipulations to modulate gene expression The transcriptional promoter and terminator sequences The ribosome-binding site and the efficiency of translation in the host organism The number of copies of the cloned gene and whether the gene is plasmid borne or integrated into the genome of the host The intrinsic stability of the cloned gene protein within the cell The final cellular location of the synthesized foreign protein Promoters Effective gene expression system requires a strong and regulatable promoter sequence upstream from the cloned gene Strong promoter has a high affinity for RNA pol Regulated promoter Some cloned DNA may produce products that are toxic to the bacterial cell, and if the promoter driving production of such gene products were not regulated, the bacterial cell might be poisoned. High levels of continual expression of a cloned gene is often detrimental to the host cell because it creates an energy drain, thereby impairing essential host cell functions. Plasmids carrying a constitutively expressed gene may be lost after several generation since cells without plasmids can take over the culture What is the best promoter type for You? Constitutive promoters always express your gene of interest and eliminate the extra complexity of adding an inducer. If you have a non-toxic gene and are not worried about the timing of your expression, using a constitutive promoter is easier. Examples of Promoters T3 T5 T7 Lac Trp 35S from CMV Chicken actin Promoter… The 35S promoter for CMV is used to express viral genes in plants because a bacterial promoter or bacteriophage promoter cannot turn on genes in plants Mammalian expression uses the strong constitutive CAG promoter that consists of the chicken actin promoter Tri Systems www.Qiagen.com A single vector is possible in three different expression systems – the T5 promoter/lac operator transcription- translation system for expression in E. coli – the p10 promoter for baculovirus-based expression in insect cells – the CAG (actin) promoter for expression in mammalian cells Regulated expression vector pTrc99A (Ref: Amann et al. Gene 69: 301-315 (1988)) Designed for the regulated expression of genes in E. coli of fused and non-fused proteins Based on pKK233-2 Origin of replication from pBR322 Has a strong hybrid promoter trp/lac The lacZ ribosome binding site (RBS) MCS of pUC18 rrnB transcription terminator Ptac or Ptrc promoter Regulated hybrid promoter containing the –35 promoter from the trp promoter and the –10 region from the lacuv5 promoter – Lacuv5 is a variant of the lac promoter that contains altered nt seq in the –10 region is stronger than the wild type lac promoter and is repressed by the lac repressor and derepressed by IPTG or lactose – Does not require activation with CAP-cAMP Lac In the absence of lactose, the E. coli lac promoter is repressed, turned off by the lac repressor protein Induction can be achieved by the addition of lactose or IPTG, prevents the binding of the repressor to the lac operator Catabolite activator protein (CAP) increases affinity of the promoter for RNA pol Trp Trp promoter is negatively regulated, turned off by a tryptophan trp-repressor protein complex that binds to the trp operator and prevents transcription of the trp operon De repression, or turning on achieved by removing tryptophan or adding 3-indole acrylic acid Consensus sequences – 5’-TATAAT-3’ for the –10 region – 5’-TTGACA-3’ for the –35 region The lacUV5 promoter has a consensus sequence at its –10 but not its –35 region The trp promoter has a consensus sequence at its –35 region but not its –10 region – By fusing the –10 region of the lac promoter and the –35 region of the trp promoter, called it the Ptac or Ptrc pTrc99A lacI gene including its corresponding promoter (PlacI). Also carries the lacIq mutation for which helps to maintain repression of Plac transcription. Produces much higher levels of the lac repressor, thereby decreasing the leakiness under non-induced conditions, i.e. transcription of a cloned gene in the absence of an inducer pTrc99A To produce the protein in pTrc99A, cells carrying the plasmid are grown to a moderate density and IPTG is added Increasing protein production Strategy used to increase protein production in recombinant E. coli involves designing an expression vector with a Ts origin of replication and a regulated promoter – Replacing the origin of replication of the plasmid pPLc2833 with that from the plasmid pKN402 – To make the plasmid pCP3 – pCP3 contains the pL promoter, the -lactamase gene for amp resistance from pLc2833 and the origin of replication from pKN402 cI repressor is present in the chromosome of the E. coli host The pL promoter is controlled by the cI repressor protein of the bacteriophage lambda, this switches off the transcription from pL promoter Cells that carry the pCP3 plasmid are grown first at 28C and then shifted to 42C At 28C the cI repressor is functional and turns off the pL promoter plasmid copy number is normal At 42C, the plasmid copy number increases and the ts cI repressor is inactivated Large scale production systems Temperature shifts take time and energy Chemical inducer e.g. IPTG is expensive Large scale production systems Two Plasmid system: In one plasmid: – The cI repressor was placed under the trp promoter (Ptrp) and inserted into a low copy plasmid In a second plasmid: – Have the cloned genes, e.g. -galactosidase and citrate synthase genes under the pL promoter Termination signals Two strong transcription terminators – T7 from E. coli bacteriophage T7 and rabbit globin terminator sequence Contains signals for the polyadenylation of the mRNA transcript – to prevent read- through transcription to ensure stability of the expressed construct Regulatory sequences Ribosome binding site (RBS) The molecular basis for differential translation is the presence of a translational initiation signal called a ribosome binding site (rbs) in the transcribed mRNA RBS Bacterial cells and human cells have different mechanisms for selecting translational start points on the mRMA This is an important consideration for producing human proteins in bacteria RBS- Bacteria Directed by a ribosome attachment sequence closely linked to the AUG initiator codon for translation. This ribosome attachment sequence consist of 6 – 8 nucleotides UAAGGAGG (Shine Dalgarno sequence) upstream of AUG This sequence is complementary to the 3’ end of the 16S ribosomal RNA, AUUCCUCC RBS Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG Bacterial mRNAs that do not have a close match to the consensus ribosome attachment sequence are not translated efficiently Generally, the stronger the binding of the mRNA to the ribosomal RNA, the greater the efficiency of translational initiation RBS - Eukaryotes The ribosome binds to an mRNA through the 5’ terminal cap structure The ribosome then scans the RNA in the 5’ to 3’ direction until it located the first AUG in the mRNA If this AUG is surrounded by a suitable sequence, translation will begin RBS - Eukaryotes This sequence in eukaryotes, called the Kozak sequence 5’-A/GCCACCAUGG which lies within a short 5' untranslated region, directs translation of mRNA Conditions required for maximum translation efficiency The RBS must be located at a precise distance from the translation start codon of the cloned gene Overproducing a human protein in E. coli requires customizing the gene to have a bacterial RBS appropriately spaced from the AUG codon for translation initiation The DNA sequence that includes the RBS through the first few codons of the gene must not contain nucleotide sequences that after transcription can fold back to form intra-strand loops, thereby blocking the interaction of the mRNA with the ribosome. Expression vector pKK233-2 An ampicillin resistance gene The tac promoter The lacZ ribosome binding site An ATG start codon located 8 nucleotides downstream from the RBS The transcription terminators T1 and T2 from lambda Copies of the cloned gene Tandem gene arrays – level of gene expression is proportional to the number of copies of the transcribed gene in the host cell STRATEGY Increase plasmid copy in the cell Clone multiple copies of the gene in tandem into a low copy number plasmid Vector Preparation Unique site in vector is digested with AvaI (C_TCGGG) Filling in with DNA polymerase I EcoRI linker (GAATT_C) inserted EcoRI linker flanked by two AvaI sites (CTCGGAATTCTCGGG) is inserted into the plasmid The gene plus the signals are cloned into the EcoRI site by digesting with AvaI to give non- sticky ends so the genes are orientated in one direction DNA integration into the host chromosome When a gene is part of the host chromosomal DNA, it is relatively stable and consequently can be maintained for many generations Factors to consider when integrating a gene the chromosome integration site must not be within an essential coding gene the input gene must be under the control of a regulated promoter for integration the DNA sequence must have some sequence homology for recombination between the two DNA molecules chromosomal site about 50 nt Integration into the host chromosome The cloned gene inserted in the middle of a cloned segment of DNA (ab) from the host chromosome on the plasmid Homologous DNA pairing occurs between plasmid-bourne DNA regions a and b and the host chromosome DNA regions a’ and b’ A double cross over event (X-X) results in the integration of the cloned gene Stability of cloned gene protein Small proteins and peptides are difficult to be stably expressed in E. coli These proteins can be stabilised by expressing them fused to a large protein such as mouse (DHFR) dihydrofolate reductase Fusion Proteins Fusion proteins are constructed at the DNA level by ligating together the coding regions of two genes One gene is the host gene that produces a stable host protein and the other gene represents the cloned gene that will produce a foreign protein When the two genes are transcribed, the foreign protein produced will be covalently linked to the stable host protein Uses of fusion protein determine its location in a cell stability of the protein used as antigens and to generate antibodies Used to purify recombinant proteins through the technique of immuno-affinity chromatography Histidine tag Fusion proteins can be designed with a His tag Can be placed at either N- or C- terminus Important that DNA is inserted in correct reading frame His tag His tag at the N-terminus most commonly used, easiest to prepare When at the N-terminus only the 5’end of the ORF must be ligated in frame When at the C-terminus the insert must be cloned in frame with the ATG start codon and the 3’ His coding system At the C-terminus His tag facilitate cloning of full length Cleavage of fusion proteins Host protein – may affect the biological functioning of the target protein – may cause allergic responses Marker Peptide Saccharomyces cerivisiae plasmid construct for the production of interleukin-2 is joined to DNA encoding a marker peptide sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) Function of Marker Peptide reduces degradation of the expressed interlukin-2 gene product and enables the product to be purified Purify recombinant proteins Monoclonal antibodies made against the marker peptide Immobilized on a polypropylene support and binds the fusion protein’s marker protein The secreted proteins are passed through the column containing the bound antibody The immunopurified fusion protein is then selectively eluted from the column marker protein is removed with bovine intestinal enterokinase. Some Fusion Protein System Fusion Partner Size Ligand Elution Condition Protein A 14kb IgG Low pH His tail 6-10aa Ni2+ Imidazole Strep-tag 10 aa Streptavidin Iminobiotin Metabolic load Increasing plasmid copy number or plasmid size Limited amounts of dissolved oxygen in the growth medium Overproduction of foreign proteins Jamming of export site Production of toxic products Overcoming O2 Limitation Use of protease-deficient strains – develop host strains that are deficient in the production of proteolytic enzymes – mutations in both the genes for the RNA pol sigma factor responsible for heat shock proteins synthesis (rpoH) and the gene for protease required for growth at high temperatures (degP) – secreted proteins had a 36-fold greater specific activity Overcoming O2 Limitation Bacterial hemoglobin Vitreoscilla bacterium, GNB obligate aerobe, normally live in oxygen poor environments such as stagnant water. synthesizes a hemoglobin-like molecule gene for Hb-like protein cloned and expressed in E. coli, the transformant displayed higher levels of protein synthesis Overproduction of foreign proteins deplete the pools of certain aminoacyl- tRNA or even certain amino acids. increase translational errors 10-fold drain the host cell of it energy diminish the usefulness of the protein may cause the protein to be immunogenic Secretion of proteins Secretion of proteins in E. coli is mediated by an N-terminal signal sequence Promote proper folding and disulfide bond formation Direct toxic proteins out of the cell Jamming of export site Over expression of protein and export from cytoplasm to cell membrane or periplasm may jam export sites thereby preventing localization of essential proteins Over expressed protein may also interfere with proper functioning of the host cell Use of His-tag at the C-terminus to prevent interferences during N-terminal processing Reference Glick and Pasternak. Molecular Biotechnology: Principles and Applications of Recombinant DNA 2nd Edition Chapter 6.
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