Manipulation of gene Expression in BacteriaEXPRESSION

EXPRESSION

EXPRESSION

BiotechnologyThe primary objective of gene cloning or genetic engineering is to express the cloned gene in a selected host organism

Host SystemBacteriaYeastInsectMammalian cells

GENETICALLY ENGINEERED PRODUCTSSome products produced by genetic engineering and are currently on the market

Bt potatoesPRV PapayaHerbicide resistant cornHuman growth hormoneInsulinhuman interferon proteinmonoclonal antibodies

Gene expression in different systems Bacterial genes being expressed in a eukaryotic systemEukaryotic genes are being expressed in bacteria

Expression vectors Vectors designed to overproduce specific gene productsUse well-characterised RNA and protein synthesis systems

Lambda ZAPIIpTrc99ApBluescript II SK+

Manipulations to modulate gene expression The transcriptional promoter and terminator sequencesThe ribosome-binding site and the efficiency of translation in the host organismThe number of copies of the cloned gene and whether the gene is plasmid borne or integrated into the genome of the hostThe intrinsic stability of the cloned gene protein within the cellThe final cellular location of the synthesized foreign protein

PromotersEffective gene expression system requires a strong and regulatable promoter sequence upstream from the cloned geneStrong 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 PromotersT3T5T7Lac Trp 35S from CMVChicken actin

PromoterThe 35S promoter for CMV is used to express viral genes in plants because a bacterial promoter or bacteriophage promoter cannot turn on genes in plantsMammalian 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 systemsthe T5 promoter/lac operator transcription-translation system for expression in E. colithe p10 promoter for baculovirus-based expression in insect cellsthe 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 proteinsBased on pKK233-2Origin of replication from pBR322Has a strong hybrid promoter trp/lacThe lacZ ribosome binding site (RBS)MCS of pUC18rrnB transcription terminator

Ptac or Ptrc promoterRegulated hybrid promoter containing the 35 promoter from the trp promoter and the 10 region from the lacuv5 promoterLacuv5 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

LacIn the absence of lactose, the E. coli lac promoter is repressed, turned off by the lac repressor proteinInduction can be achieved by the addition of lactose or IPTG, prevents the binding of the repressor to the lac operatorCatabolite activator protein (CAP) increases affinity of the promoter for RNA pol

TrpTrp 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 operonDe repression, or turning on achieved by removing tryptophan or adding 3-indole acrylic acid

Consensus sequences5-TATAAT-3 for the 10 region5-TTGACA-3 for the 35 region

The lacUV5 promoter has a consensus sequence at its 10 but not its 35 regionThe 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

pTrc99AlacI 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

pTrc99ATo produce the protein in pTrc99A, cells carrying the plasmid are grown to a moderate density and IPTG is added

Increasing protein productionStrategy used to increase protein production in recombinant E. coli involves designing an expression vector with a Ts origin of replication and a regulated promoterReplacing the origin of replication of the plasmid pPLc2833 with that from the plasmid pKN402To 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 promoterCells that carry the pCP3 plasmid are grown first at 28C and then shifted to 42CAt 28C the cI repressor is functional and turns off the pL promoter plasmid copy number is normalAt 42C, the plasmid copy number increases and the ts cI repressor is inactivated

Large scale production systemsTemperature 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 plasmidIn a second plasmid:Have the cloned genes, e.g. -galactosidase and citrate synthase genes under the pL promoter

Termination signalsTwo strong transcription terminators T7 from E. coli bacteriophage T7 and rabbit globin terminator sequenceContains 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

RBSBacterial cells and human cells have different mechanisms for selecting translational start points on the mRMAThis is an important consideration for producing human proteins in bacteria

RBS- BacteriaDirected 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

RBSActivity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUGBacterial mRNAs that do not have a close match to the consensus ribosome attachment sequence are not translated efficientlyGenerally, the stronger the binding of the mRNA to the ribosomal RNA, the greater the efficiency of translational initiation

RBS - EukaryotesThe ribosome binds to an mRNA through the 5 terminal cap structureThe ribosome then scans the RNA in the 5 to 3 direction until it located the first AUG in the mRNAIf this AUG is surrounded by a suitable sequence, translation will begin

RBS - EukaryotesThis 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 geneOverproducing a human protein in E. coli requires customizing the gene to have a bacterial RBS appropriately spaced from the AUG codon for translation initiationThe 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 promoterThe lacZ ribosome binding site An ATG start codon located 8 nucleotides downstream from the RBSThe 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

STRATEGYIncrease plasmid copy in the cellClone multiple copies of the gene in tandem into a low copy number plasmid

Vector PreparationUnique site in vector is digested with AvaI (C_TCGGG)Filling in with DNA polymerase IEcoRI linker (GAATT_C) inserted EcoRI linker flanked by two AvaI sites (CTCGGAATTCTCGGG) is inserted into the plasmidThe 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 genethe input gene must be under the control of a regulated promoterfor integration the DNA sequence must have some sequence homology for recombination between the two DNA moleculeschromosomal 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 plasmidHomologous DNA pairing occurs between plasmid-bourne DNA regions a and b and the host chromosome DNA regions a and bA double cross over event (X-X) results in the integration of the cloned gene

Stability of cloned gene proteinSmall proteins and peptides are difficult to be stably expressed in E. coliThese 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 genesOne gene is the host gene that produces a stable host protein and the other gene represents the cloned gene that will produce a foreign proteinWhen the two genes are transcribed, the foreign protein produced will be covalently linked to the stable host protein

Uses of fusion proteindetermine its location in a cellstability of the protein used as antigens and to generate antibodiesUsed to purify recombinant proteins through the technique of immuno-affinity chromatography

Histidine tagFusion proteins can be designed with a His tagCan be placed at either N- or C- terminusImportant that DNA is inserted in correct reading frame

His tagHis tag at the N-terminus most commonly used, easiest to prepareWhen at the N-terminus only the 5end of the ORF must be ligated in frameWhen at the C-terminus the insert must be cloned in frame with the ATG start codon and the 3 His coding systemAt the C-terminus His tag facilitate cloning of full length

Cleavage of fusion proteins Host protein

may affect the biological functioning of the target proteinmay cause allergic responses

Marker PeptideSaccharomyces 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 Peptidereduces degradation of the expressed interlukin-2 gene product andenables the product to be purified

Purify recombinant proteinsMonoclonal antibodies made against the marker peptide Immobilized on a polypropylene support and binds the fusion proteins marker proteinThe secreted proteins are passed through the column containing the bound antibodyThe immunopurified fusion protein is then selectively eluted from the columnmarker protein is removed with bovine intestinal enterokinase.

Some Fusion Protein SystemFusion Partner SizeLigand Elution ConditionProtein A14kbIgGLow pHHis tail6-10aaNi2+ImidazoleStrep-tag10 aa Streptavidin Iminobiotin

Metabolic loadIncreasing plasmid copy number or plasmid size Limited amounts of dissolved oxygen in the growth medium Overproduction of foreign proteinsJamming of export site Production of toxic products

Overcoming O2 LimitationUse 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 LimitationBacterial 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-folddrain the host cell of it energy diminish the usefulness of the protein may cause the protein to be immunogenic

Secretion of proteinsSecretion of proteins in E. coli is mediated by an N-terminal signal sequencePromote proper folding and disulfide bond formation Direct toxic proteins out of the cell

Jamming of export siteOver expression of protein and export from cytoplasm to cell membrane or periplasm may jam export sites thereby preventing localization of essential proteinsOver expressed protein may also interfere with proper functioning of the host cellUse of His-tag at the C-terminus to prevent interferences during N-terminal processing

ReferenceGlick and Pasternak. Molecular Biotechnology: Principles and Applications of Recombinant DNA 2nd Edition Chapter 6.