* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Factors that influence gene expression
Histone acetylation and deacetylation wikipedia , lookup
Epitranscriptome wikipedia , lookup
Magnesium transporter wikipedia , lookup
Molecular evolution wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Gene expression profiling wikipedia , lookup
Secreted frizzled-related protein 1 wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Western blot wikipedia , lookup
Point mutation wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Protein adsorption wikipedia , lookup
Protein moonlighting wikipedia , lookup
Gene regulatory network wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Transcriptional regulation wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene expression wikipedia , lookup
Chap 5 Manipulation of Gene Expression in Procaryotes I. Introduction A major objective of gene cloning is the expression of the cloned gene to study the biologic functions or to produce recombinant proteins (i.e. insulin). But gene cloning doesn’t guarantee successful expression. Factors that influence gene expression 1. The nature of the transcriptional promoter and terminator sequences 2. The strength of ribosome binding site 3. Efficiency of translation (mRNA stability, mRNA secondary structure..) 4. # of the cloned gene copies (or # of plasmids) and whether the gene is plasmid borne or integrated into the chromosome. 5. Nature and cellular location of the expressed protein (intra- or extracellular? secreted? toxic?) 6. Post-translational processing: glycosylation, proteolytic processing… 7. The intrinsic stability of the protein (misfolding of the proteins? susceptible to proteolysis?) A large fraction of proteins (varying from 30% to 70% of all proteins made) is immediately degraded after synthesis before forming functional proteins8. These socalled DRiPs (defective ribosomal products) are the result of defective transcription or translation, alternative reading frame usage, failed assembly into larger protein complexes, the incorporation of wrong amino acids owing to mistakes by aminoacyl-tRNA synthetases or altered ubiquitin modifications. DRiPs are immediately degraded to prevent the formation of protein aggregates, which would affect cell viability. Ubiquitination is a post-translational modification in which ubiquitin, a 76–amino acid protein, is covalently added to lysine residues. In humans, the ubiquitination reaction is catalyzed by >500 E3 ligases, each of which transfers ubiquitin 1 to specific protein targets. There are several types of ubiquitin modification, and these may have different effects on target proteins. The best known is the polyubiquitin chain, which targets proteins for proteasomal degradation. The polyubiquitin chain begins with a ubiquitin conjugated at its C terminus to a lysine residue in a target protein. 26S proteasome: A giant multicatalytic protease that resides in the cytosol and the nucleus. The 20S core, which contains three distinct catalytic subunits, can be appended at either end by a 19S cap or an 11S cap. The binding of two 19S caps to the 20S core forms the 26S proteasome, which degrades polyubiquitylated proteins into peptides1. Some of the above factors can be improved by proper design (i.e. select strong promoter, use multiple gene copies…) Choice of expression system is very important!!! Major expression systems are classified into procaryotic and eucaryotic. Procaryotic (e.g. E. coli): Pros: 1. Very well-studied and common in protein production. 2. Grow fast (doubling time20 min), grow easily easy fermenter operation. 3. Normally high yield (high cell density). 4. Minimum media (simple composition, e.g. Na+, K+, Mg2+, Ca2+, NH4+, Cl-, SO42-, glucose and carbon sources) cheap. Cons: 1. Often fail to perform suitable post-translational modifications. 2. Inclusion body (insoluble proteins) when overexpressing makes purification and regaining of protein conformation (protein renaturation) more difficult. Eucaryotic: next chapter II. Strong and regulatable promoters 1 Why strong promoters? Some endogenously expressed proteins (e.g. viral proteins or tumor proteins) are degraded to short peptides and routed to MHC class I in ER, where the formed complex leaves ER to the surface for presentation. 2 Has higher affinity for RNA pol so the downstream gene is highly (frequently) transcribed. Why regulatable promoters? Continuous overexpression of a cloned gene is often detrimental to the host cell because it drains the energy and other resources and impair cellular functions. genes are constructed under strong and regulatable promoters. genes are expressed only when “induced”. Examples 1. E. coli lac promoter: (a) Regulated by IPTG Cells are grown in the absence of lactose and repressor binds to the operatorgenes can’t be transcribed. Only when IPTG is added then starts the gene expression. Lac repressor promoter operator Gene 1, 2,3,.. Induction (turn on) by IPTG (isopropyl--D-thiogalactopyranoside) IPTG prevents lac repressor from binding to the operator Transcription occurs Very common (b) regulated by CAP (catabolite activator protein) 3 cAMP CAP promoter operator Gene 1, 2,3,.. Binding of cAMP to CAP further enhances the affinity for RNA pol *level of cyclic AMP is highest when glucose level is low cAMP CAP promoter operator Gene 1, 2,3,.. RNA pol Combining the above, induce protein expression at high IPTG (or lactose) and low [glucose]. (high [cAMP])highest transcription. 2. Trp promoter: (regulates the transcription of genes responsible for Trp synthesis) off (negatively regulated): tryptophan-trp repressor protein complex binding to trp operator transcription shutdown 3. on (positively regulated): removal of tryptophan Bacteriophage T7 promoter: T7 RNA pol IPTG (to T7 promoter Target gene lac promoter T7 RNA pol gene induc T7 promoter is very strong, but requires T7 RNA pol to activate. e Two recombinant genes can be co-introduced into the cells for expression. Alternatively, the genes encoding T7 RNA pol can be integrated into the chromosomal DNA to form a stable cell line. 4. pL promoter (from bacteriophage ): 4 Controlled by cI repressor protein Cells carrying temp-sensitive cI repressor are grown at 28C (cI repressor is expressed under its own promoter pCI at 28C) cI repressor prevents transcription when CD is high enoughincrease to 42C thermosensitive cI repressor is inactivated transcription is on. Effectiveness of deactivating a repressor depends on # of repressor # of copies of promoter sequences ratio too large difficult to induce ratio too small transcription is “leaky” (transcription occurs in the absence of inducer) Strategy: Put repressor genes in a plasmid: low copy # (e.g. 1-8 copies/cell) Put promoter-target gene in another plasmid: high copy number (e.g. 30-300 copies/cell) maintain the ratio to effectively deactivate and activate. III.Expression vectors Regulatable, strong promoters may not guarantee high yield of gene products. Efficiency of translation, stability of protein, etc. also are factors2. Expression vectors are similar to cloning vectors but contain more elements to confer efficient expression. e.g. The expression plasmid pKK233-2 contains: tac promoter (a hybrid that includes the -10 region of lac promoter and -35 region of trp promoter, can be induced by IPTG, 3X and 10X stronger than trp and lac promoters, respectively) RBS, ori. (RBS: a sequence of 6-8 nt (e.g. UAAGGAGG) in mRNA that can base pair with rRNA on the ribosome, generally, binding of mRNA to rRNA increases, the translation initiation increases) 2 Not all mRNA are translated in the same efficiency, differential translation and transcriptional regulation enable the cells to adapt to different stresses (environmental, heat shock, oxygen…) 5 An ATG start codon about 8 nt downstream from the RBS (optional) Multiple cloning site Ampr gene as a selectable marker Note: the RNA sequence from RBS to the first few codons of the cloned gene must not form intrastrand loops, which hampers the binding to ribosome DNA sequence is written as the coding strand, so ATG is often seen as the starting point. IV. Fusion Proteins Problems: yield of foreign proteins normally low for various reasons (e.g. degradation by proteases) Solution: covalently attach the cloned gene product to a stable (host) protein to form a fusion protein to protect the desired recombinant protein. Construct at DNA level transcribed RNA must have correct base sequence (stop codon in the middle must be eliminated) Reading frame must be correct, base sequence in the linker must be precise, otherwise ORF will be wrong (need to know the precise sequence of these two proteins) Cleavage of fusion proteins The fusion may not be suitable as the final product because: The biological function might be lost 6 Stringent regulation by government agencies (e.g. FDA) The EK cleavage site enables the cleavage of the fusion by enterokinase at the specified site. Another linker often used is the Xa linker (Ile-Glu-Gly-Arg) which can be recognized by a blood coagulation factor (Xa) and specifically recognized at the C-terminus the desired protein should therefore be in the second segment. Applications of fusion proteins (many applications, give 2 example only) 1. Simplifying purification dual function of the fusion: reduce the degradation, enable the cleavage Flag (a peptide recognized by EK) -Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-IL2 IL2: a cytokine that stimulates both T-cell growth and B-cell Ab synthesis enable the product to be purified by immunoaffinity chromatography in which MAb directed against Flag is immobilized on a polypropylene support and used as a ligand to bind the fusion. 2. Stabilizing the protein (e.g. Enbrel) Enbrel is a recombinant protein that is approved by FDA to treat autoimmune diseases (e.g. rheumatoid arthritis and psoriatic arthritis) by interfering with tumor necrosis factor (TNF; a soluble inflammatory cytokine) by acting as a TNF inhibitor. TNF- is the "master regulator" of the inflammatory response in many organ systems and excess TNF causes aberrant inflammation. Enbrel is a fusion protein produced by recombinant DNA. It fuses the TNF receptor 2 to the Fc end of the IgG1 antibody. TNF receptor 2 binds to TNF-. The protein is highly active and unusually stable as a modality for blockade of TNF in vivo. V. Golden Gate Shuffling: A One-Pot DNA shuffling Method Limitations of the traditional cloning methods 7 Time consuming Inefficient Golden Gate Shuffling is a protocol to assemble separate DNA fragments together into a vector in one step and one tube. The principle of the cloning strategy is based on the ability of type IIS restriction enzymes (e.g. BsaI) to cut outside of their recognition site. Two DNA ends terminated by the same 4 nucleotides (sequence f, composed of nucleotides 1234) can be synthesized by PCR, where sequences f are flanked by a BsaI recognition sequence, B. The type IIs restriction enzymes removes the enzyme recognition sites and generates ends with complementary 4 nt overhangs. These ends can be ligated seamlessly, creating a junction that lacks the original site. Ex: One-pot one-step assembly of 9 fragments First select a number of 4 nucleotides ‘recombination sites’ on a nucleotide sequence alignment of several homologous genes. The selection of these recombination sites defines modules that consist of a core sequence (C1-C9) flanked by two 4 nt sequences. These 9 modules can be amplified by PCR with primers designed to add flanking BsaI sites on each side of the modules (the BsaI cleavage sites perfectly overlapping with the recombination sites) and cloned into 9 plasmids separately. 8 The recipient expression vector, pX-LacZ contains two BsaI sites compatible with the first (C1) and last (C9) modules. Mix the 9 module plasmids and 1 recipient plasmid into one tube. Add BsaI and ligase. VI. Increasing protein stability Normally, the half lives of proteins range from a few minutes to hours (some exceptions exist, e.g. collagen has a half life of years). Normally, proteins with more disulfide bonds (S-S between Cys) and certain amino acids at the N-terminus are more stable more proteins accumulate and the yield increases. Ex: stability of -galactosidase with certain a.a added to the N-terminus Strategies: Change the a.a. at the N-terminus Increase the number of S-S bonds Co-express chaperone proteins (e.g. groEL, dna J, dna a.a. added Half life Met, Ser, Ala > 20 h Thr, Val, Gly > 20 h Ile, Glu > 30 min Arg 2 min K….) to aid the protein folding VII. Overcoming O2 Limitation Oxygen is generally required for cell growth, to support respiration and maintain cellular functions and protein expression, but oxygen’s solubility is low. If the CD is high, even larger amount of air or oxygen or increasing the stirring speed may not be enough. When O2 depletion occurs, cells would enter stationary phase and die eventually. If engineering approaches fail, what can we do? bacterial hemoglobin 9 Solve: bacterium “Vitreoscilla” inhabits in stagnant ponds (oxygen deficient). To obtain oxygen for growth and metabolism, the bacteria express a hemoglobin-like protein that fetch oxygen from the environment and transport into the cells. When this gene is cloned and expressed in E. coli, the recombinant E. coli shows higher metabolic activity and higher protein production at low levels of O2. VIII. DNA Integration into the Host Chromosome Why integrate DNA into the chromosome? Plasmid-borne expression drains the cellular energy because the antibiotics-resistant and other genes are expressed and the plasmid replication requires the resources and energies too. Plasmid instability: plasmid-free cells outgrow plasmid-bearing cells, so after several passages, the percentage of cells bearing plasmids dropsprotein expression level drops. Integration using the plasmid 1. Choose a suitable integration site. 2. Clone part of the chromosomal DNA sequence at the integration site into the vector (e.g. plasmid). The chromosomal DNA sequence on the vector and at the integration site must be similar in sequence, typically >500 bp, so that homologous recombination can occur. 3. Clone the target gene (and promoter) into the plasmid (vector) flanked by the chromosomal DNA sequence. 4. Transfer the plasmid into a host cell (The vector does not replicate or can be removed from the host cell). 5. Select the host cells that have the target gene integrated into the chromosome. 6. Drawback: inefficient, long homology arm is required. 10 Integration using the red system [1]: Recombineering Derived from bacteriophage λ, requires 3 proteins: Exo, Beta, and Gam 1. Exo has 5’ exonuclease activity that degrades one entire strand of dsDNA to ssDNA when dsDNA is introduced into a cell. The ssDNA is stabilized and protected from exonuclease attack when Beta binds to it. Beta also delivers ssDNA to the target replication fork and facilitates annealing of the ssDNA to the target site (the mechanism is proposed but is not confirmed and still controversial). Gam in E. coli is to inhibit the activity of the bacterial RecBCD protein complex by binding to it (otherwise RecBCD would degrade the incoming ds or ss DNA) 2. These (Red) proteins should be tightly regulated because continuous expression of Exo and Beta increases background recombination and long-existing Gam could be toxic to the cell. 11 3. Transform a plasmid encoding Exo, Beta, and Gam under an inducible promoter (e.g. pL or others) Transiently induce the Red proteins expression Introduce the template DNA (usually electroporation of oligonucleotides or PCR products) with the homology arm (as short as 50 bp)recombination. 4. red system is widely used for singleplex and multiplex genome engineering. With ss DNA, the recombination efficiency can be up to 25%. With dsDNA, the efficiency is 0.2%. 5. Pros: The red system significantly improves the recombination efficiency The homology arm can be as short as 50 bp, thus the gene can be amplified by PCR with flanking homology arms 6. Cons: The system is not suitable for inserting long DNA Increasing Secretion Secretion is important for many human proteins (e.g. adrenaline, growth factors and many other blood proteins are secreted). 12 In industry, it’s often desired that the proteins be secreted because: secreted proteins tends to be more stable. For example, a recombinant proinsulin is 10X more stable if exported into the periplasm (the space between the inner and the outer membrane) . Secreted proteins may give higher purification recovery yield because they are free from thousands of cellular proteins. Drawback: recombinant protein concentration in the medium is low. Secreted proteins have a signal peptide at the N-terminus, facilitating the protein transport though the secretory pathway. When crossing the membrane, the signal peptide is cleaved by peptidase to become the mature protein. Problem: E. coli and other Gram negative bacteria have outer membranes, which prevent proteins from secreting to the medium. Solve: Use gram-positive or eucaryotes which do not have outer membranes (but Gram-negative bacteria such as E. coli is usually excellent first-choice). Fuse a signal peptide or engineer a fusion protein with signal peptide at the Nterminus. Lower the expression level because sometimes over-expression could overwhelm the secretion machinery, thus mitigating the secretion. Co-express a limiting factor in the secretion pathway. For instance, clone prl A4 and secE genes (which encode the major components of the molecular apparatus that moves proteins across the membrane) into E. coli % of secreted (and mature) protein increases from 50% to 90%. Clone bacteriocin release protein: bacteriocin (in Gram negative bacteria) is secreted with the help of bacteriocin release protein, which permeabilize the inner and outer membranes Co-express this protein with the target protein. 13 X. Reducing the Metabolic Load Expression of foreign gene often changes the metabolism and impairs the normal cellular function, due to the increased metabolic load (burden) for the following reasons: Competing for amino acids, tRNA and energy (ATP). DO is often insufficient for cell metabolism and plasmid maintenance. Increasing plasmid copy number often requires increasing amounts of cellular energy for plasmid replication and maintenance. Foreign proteins may jam the export sites and impair proper localization of host proteins. Foreign proteins may be toxic to the cells. Outcomes: Plasmid instability: cells w/o plasmid outgrow cells with plasmid loss of recombinant plasmid. Energy intensive processes such as nitrogen fixation and protein synthesis slow down. Translational error: because tRNA could be limiting so incorrect a.a. may be incorporated (chance could be 10 times more when overexpression occurs). Solution: Use low copy # plasmid instead of high copy number plasmid. Integrate the foreign DNA into the chromosome. Use strong, regulatable promoter, so cell culture is divided into two phases: Growth phase: cell growth without target protein expression Production phase: when CD is high, production is induced (e.g. by IPTG, heat…) Express at a modest level (e.g. 5% of the total protein), but at high CD in fermentation. 14 XI. Appendix Biopharmaceutical Market 15 Aggarwal, S. What’s fueling the biotech engine-2012-2013. Nat. Biotechnol. 2014: 32-39 16 17 Inclusion body (IB) Protein aggregates that usually lack biologic functions separate the IB by centrifugation or filtration (may facilitate purification)IB denaturation then renaturation. Denaturation Performed by strong acid, strong base, high temperature and pressure, proteases etc. Usually chemical agents are used: Urea: 8-10 M, destroy H-bond and hydrophobic interaction. Guanidine hydrochloride (GuHCl): 6-8 M, disrupts hydrophobic and ionic interactions. Dithiothreitol (DTT), -mercaptomethanol: disrupt the S-S bond. EDTA or EGTA: chelate metal ions to avoid unwanted chemical reactions. Renaturation (refolding) Dialysis: change the buffer and dilute the denaturant concentration gradually. Renaturation buffer: usually contains Tris-HCl (pH buffer), low concentration of denaturant (e.g. urea) to prevent aggregation and oxidizing agent to oxidize the –SH group for S-S bond formation. 參考文獻 [1] Jeong J, Cho N, Jung D, Bang D. Genome-scale genetic engineering in Escherichia coli. Biotechnology Advances 2013; 31:804-10. 18