BL5602-L3 Recombinant DNA 2.pdf

How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene wi...

How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected vector (recombine DNA) 3. Delivery of recombinant DNA into a host cell. 4. Selection of genetically modified host 5. Breeding for offspring with desired genetic modification DNA cloning Recombinant protein production, desirable phenotype Link target DNA to vector- recombine DNA Recombination cloning – Restriction enzymes and ligase EcoRI HindIII HindIII EcoRI Restriction sites may be present in the gene fragment.  Integration occar at the att site a via Homologous Recombination lambda o Catalyse by encyme Integrase : Bacterial Emzyme Recombination cloning – Gateway cloning Rapid cloning without restriction enzymes and ligase, 1 hr INT – integrase ccdB codes a toxim XIS – excisionase Accurate results, >95% efficiency that can kill the bacteria Particularly useful to clone multiple aUse the gene of Ihferest So the phage gehome DNA fragments into the same vector to replace the toxin gene can Integrate & revease Attachment (att) sequence (< 50 bp) cpopout ) of the host can be conveniently added by PCR genome ccdB encodes a toxin (gyrase inhibitor) excisionase catalyze that kills bacteria unless it contain an the reverse process antitoxin gene WT V法ctor opon integration BP is comverted to LR LR reaction. Fast excision convert LR to BR between attL and attR BOB’=Bacteria POP’=Phage BP/ LR complehe BOP’ POB’ X BOB’ POP’ X. in 5 minule toxin INT + XIS toxin towoe2 o yome o omultiple Fragment in Con : INT. Gcar BP reaction Background gtowth between attB and attP. sive Select AmpR colonies. Enuyme expen 2 step cloning might be usefuliFyou want to Intooclece : o Read to Understand - more , ditferend 1 moltiple genes into the Same Vector 走  Rapid cloning of a DNA fragment to the Gateway vector by PCR linterest Gene of INT + XIS Spectinomycin selection Introduce att seuences into the Vector using PCR to save fime Example of att sequences P region O region B’ region B region O region P’ region Fu et al (2008) Rapid one-step recombinational cloning. Nucleic Acids Research 36:e54  multiple orerlapping Recombination cloning – Gibson assembly Can join sequence together USing Restriction ERyme or Need PCR primer - ☆ with Up to 15 fragments can be Gateway Requires Restriction overlap sequences. combined simultaneously site oz att site not divect comnection so its No restriction site scar remains θ Explain this step between two DNA fragments The Gibson assembly method sirngle hounded ' 3 overhangs can also be used for site directed mutagenesis to incorporate site- single strand specific mutations such as anneal insertions, deletions, and point 0 mutations From 3to 51 EXonule ase { DNA DNA Poly merase Ligase This methodis still patented - need to pay For commercial use 一 Recombination cloning – Gibson assembly  Recombination cloning – Gibson assembly Can join as many modules as you like into your own rector Promoter Gene-of-interest Origin of Replication Antibiotics resistance Epitope tags etc Gene delivery methods depend on the recombinant DNA and host cells Physical method. Biological method Chemical method Gene delivery methods depend on the recombinant DNA and host cells Chigher EFFiciency ) Larger amount = o 1 Cells into the Cells shot the material Gene Gun: mainly used in plants. Very laboun intensive Microinjection: inject genetic materials (e.g. & time consuming DNA, RNA) into cells (animal eggs). mainly animalCells https://www.thermofisher.com/th/en/home/life-science/cell-culture/transfection/methods.html Gene delivery methods depend on the recombinant DNA and host cells https://www.thermofisher.com/th/en/home/life-science/cell-culture/transfection/methods.html  Gene delivery examples -- bacteria Transformation: A naturally-occurring process where external DNA is picked up by some bacterial cells. The classic Griffith’s experiment leading to the discovery of DNA as genetic material. It is also the first time transformation was observed in bacteria. Bacteria can take up Foreign DNA as long as they have the right Receptor https://www.youtube.com/watch?v=MRBdbKFisgI Ways to enhance transformation natural transformation is not efficient metal lon - Positively Charged membrane is - negatively Charged.  High-efficiency transformation bacterial open up membrane 一  Gene delivery examples -- bacteria Transduction: using Phages A naturally-occurring process in which a virus transfers genetic material from one bacterium to another.  Gene delivery examples -- bacteria Transduction: A naturally-occurring process in which a virus transfers genetic material from one bacterium to another. Methods to introduce recombinant DNA into host cells How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected vector (recombine DNA) 3. Delivery of recombinant DNA into a host cell. 4. Selection of genetically modified host 5. Breeding for offspring with desired genetic modification Drug selectable markers/ recombinant protein production/ desirable phenotype Expression of recombinant proteins in microbial and cell systems 1. Various popular expression systems for production of recombinant proteins (Main) Bacteria Yeast Insect cells Mammalian cells 2. Genetic engineering of biopathways 3. Synthetic bacteria and yeast } synthetic Biology Basic requirements for protein expression 1. Gene construct for protein expression 2. Host cells Bacteria (E. coli, Bacillus subtilis) Yeast (Saccharomyces cerevisiae) Insect cells Mammalian cells (CHO) Basic elements of gene construct Promoter Structural Gene Terminator Expression level mRNA Inducibility Transcriptional Protein termination  Escherichia coli (E. coli) Easy to grow in liquid and on agar Fast growing, doubling time 20 min in log phase Simple in structure and fewer genes (~4400 genes) Stored at -70oC for long term Well studied and excellent research resources or infrastructure Feasible to scale up for biotechnology production 1982, the first licensed human drug, insulin, produced by recombinant DNA expression in E. coli (Humulin) was FDA approved and marketed.  Bacillus subtilis Gram positive probiotic (“good” bacteria) 4100 genes Can form hard spores to survive indefinitely Production of secreted proteins through the EM picture of cross section single membrane Easier for large scale of production and often used as the final host for industrial production single Bacillus subtilis membrane E. coli two E. coli membrane Basic elements of protein expression construct Promoter Structural Gene Terminator Expression level mRNA Inducibility Transcriptional Protein termination Bacterial gene expression Basal and constitutional promoter with strong transcriptional activity Dolymerase Binding Disadvantages of bacterial expression: 1. Strong constitutional expression may be detrimental to the host (e.g. energy drain) 2. Plasmid may be easily lost without chemical selection  Inducible expression system: lac promoter (LacI) + IPTG (isopropyl-β-D-1- CAP, Catabolite activator thiogalactopyranoside) protein, aka cAMP receptor protein Two phases of bacterial culture: 1. Growth phase without transcription of targeted gene 2. Induction phase (+IPTG and low glucose) with induced expression of targeted gene lactose/IPTG removes lac repressor Low glucose increases cAMP No inducer , no transcription  Example of expression vector pKK233-2 Features: tac promoter (a hybrid from two promoters, trp and lac, just behave like lac but stronger) Ampicillin resistant gene (Ampr) LacZ ribosome-binding site (rbs) Three cloning sites (NcoI, PstI and HindIII) Two transcription termination sequences (T1, T2) Factors to consider for expression of recombinant proteins Regulation of transcription Increasing translation efficiency Increasing protein secretion § Secretion into periplasm § Secretion into the medium Facilitating protein purification § Expression of fusion protein  Increasing translation efficiency 1. Ensure exposure of ribosome binding site and AUG initiation \ codon 2. Reduce RNA secondary structure in general 3. Codon optimization: using highly used codons in E. coli. Biasan Codon do Codon Opfimisaton Ribosome binding site Example of RNA secondary structure that would prevent efficient translation. Molecular Biotechnology 5th edition Release Factor instead will bind OF tRNA Molecular Biotechnology 5th edition Protein Production Intracellular: Need to disrupt cells after fermentation and cell harvesting More purification steps are required Extracellular: Secreted directly into the medium No need to break the cells to release the recombinant protein Fewer steps of purification needed Secreted recombinant protein to the periplasm or to the medium E. coli engineered to secrete a foreign protein to the periplasm by using a secretion signal send the protch to the periplasm space o prevent degradation. Break Bacteriocin release factor membrane - activates phospholipase A, which is present in the bacterial inner membrane, and cleaves membrane phospholipids so that both the inner and outer membranes are permeabilized. Gene fusions for expressing recombinant proteins in heterologous hosts Stabilizing recombinant proteins: Foreign proteins are often rapidly degraded by the host proteases; this may sometimes be avoided by gene fusion strategy Facilitating purification: Allow rapid and efficient purification based on the properties of fusion partner Localization of recombinant proteins: Proteins can be localized to different compartments of the host cells through specific protein/peptides fused to your protein of interest A number of different affinity-fusion systems have been developed (e.g. GST, His tag) Gene fusion strategies k Common fusion strategies are N-terminal and C-terminal fusions. Fusion partner A encodes an affinity tag will also facilitate purification: N-terminal fusion to your Protein of Interest (X) A X P T Terminator C-terminal fusion to your Protein of Interest (X) P T X A Fusion partner A (affinity tag) promoter (X+A= Fusion protein) Molecular Biotechnology 4th edition Expression of GST fusion proteins Glutathione-S-Transferase (GST), a 26 kDa cytoplasmic protein, can be fused to either the N- or C- terminal. GST-fusion proteins are soluble, easily purified from lysed cells under non- denaturing conditions by using glutathione- agarose beads, followed by elution in the What is the purpose to presence of reduced glutathione include the LacI gene ? High yield and most fusion proteins remain soluble unlike other highly expressed recombinant proteins in E. coli GST portion can be removed by a sequence- specific protease like thrombin after purification Advantages and Disadvantages of bacterial expression Advantages: Cultured in large quantities in a short time (E. coli doubles in 20 min in log phase) Fermentation methods are well established Microorganisms can be subjected to genetic manipulation more readily than animals and plants Disadvantages Sometimes intracellular accumulation of recombinant protein with insoluble inclusion bodies Unable to undertake posttranslational modifications such as glycosylation, phosphorylation and specific proteolytic cleavages Limited ability to carry out extensive disulfide bond formation Adverse public perception of products produced by recombinant methodologies Molecular Biotechnology 4th edition  Yeast (Saccharomyces cerevisiae ) 16 chromosomes with telomeres and centromeres and ~6,000 genes Allows extrachromomal DNA, e.g. 2-micron circle plasmid Well studied in molecular biology and genetics Organelles, e.g. mitochondria, ER, Golgi apparatus, etc. capable of some posttranslational modifications Easy to culture Fast growing, doubling time, 90 min Can be stored at -70oC for long term Antient biotechnology tool and well established in biotechnology industry Budding yeast  Yeast expression system Easy to Introduce High, stable plasmid in yeast GAPD (glyceraldehyde-3- phosphate dehydrogenase) Essential gene for termination site leucine biosynthesis and used for plasmid selection; without the (gene of interest) plasmid, yeast can not survive in medium lacking leucine. SOD (superoxide dismutase) ori Y Constitutive promoter Derived from bacterial plasmid and For bacterial amplification and selection 一一一一  Advantages and Disadvantages of yeast expression Advantages: Well established fermentation technology for scale-up production Ability to carry out post-translational modifications of recombinant proteins no safety ( ssue Proven history of use in many biotechnology processes and most are GRAS (Generally Regarded As Safe) listed by FDA as food ingredient Disadvantages: Recombinant proteins usually expressed at low levels Retention of expressed protein in the periplasmic space Adverse public perception of products produced by recombinant methodologies 0 thick cell wall -Challenges associated with Purification 一 Molecular Biotechnology 4th edition  Baculovirus-insect cell expression system Baculovirus are specific to arthropods including insects Polyhydrin (non-essential gene) promoter is exceptionally strong, producing 25% mRNA in infected cells viral only infeot cells tm no threat to human , o use host DNA AcMNPV: Autographa californica multiple nucleopolyhedrovirus Polymerase Xother elements in translation & transcription. Once InFected , Insect Cells own protein production stops , Oers et al (2015) Thirty years of baculovirus–insect cell protein expression… J Gen Virol 96:6-23.  Baculovirus-insect cell expression system https://www.creativebiomart.net/baculovirus-insect-cell-expression-systems.htm Tn7, bacterial transposon Helper for sequence transposase Bacmid, a very large plasmid containing the modified baculovirus genome Main advantages: High expression Low cost Good solubility Sf9 insect cells, derived from Good PTM (post- fall armyworm (Spodoptera translational Large, round and frugiperda) ovarian tissue, can modification) with granules be cultured serum-free, attached or in suspension. Baculovirus-insect cell expression system High protein yield expression system due to the strong polyhedron gene promoter (25% mRNA produced in the cell) Molecular Biotechnology 5th edition Mammalian cell expression systems Fast mammalian cell expensive X Does not replicate as cuture are Currently about half of the commercially available therapeutic proteins (e.g. antibodies) are produced from mammalian cells CHO (Chinese hamster ovary) cells are the most commonly used. o Human-like glycans o Serum-free, high-density suspension culture o high yield of heterologous proteins o Long term stable expression o Free of many pathogens (Easier to get approval as many virus including HIV, influenza, polio, herpes and measles do not replicate in CHO)  Mammalian cell expression systems Generalized mammalian expression vector multiple cloning site selectable marker gene P, promoter; I, intron enhancer (not essential); pa, polyadenylation; TT, termination of transcription Kozak sequence (K), signal sequence (S), protein affinity tag (T), proteolytic cleavage site (P) stop codon (SC) Two-gene expression vector  Jayapal et al (2007) Sometimes intracellular accumulation of recombinant protein with insoluble inclusion bodies. CHO Almost entirely therapeutic agents. Consortium (https://www.aiche.org/sites/default/files/docs/pages/CHO.pdf)  Intended use of E. coli E. coli expression is generally a first choice for expression of any protein. The likelihood of success is greatest when the protein is small (preferably less than about 70 kDa) Does not have unpaired cysteines or more than two or three intra-molecular disulfide bonds Does not require post-translational modification for activity or solubility Why to express in eukaryotic cells? Some eukaryotic proteins made by bacteria are unstable or inactive, especially those proteins requiring post-translational modifications. 一一  Comparison of different host systems for production of recombinant proteins Parameter Bacteria Yeast Mammalian Transgenic Transgenic cell culture plants animals (milk) Glycosylation None Incorrect Correct Generally Correct correct, small difference Multimeric Limited Limited Limited Yes proteins assembled effective Production Low-medium Medium High Very low low costs Protein Low Medium High High folding accuracy Protein yield High Medium-high Low-medium Medium Scale-up High High Low Very high Very high capacity Scale-up cost High High High Low Low Time required Low Low-medium Medium-high High High Skilled Medium Medium High Low Low workers required Acceptable to Yes Yes Yes Not yet Yes regulators Bertolini et al (2016) The transgenic animal platform for biopharmaceutical production. Transgenic Res. 25:329-343. Summary Cloning methods Various popular expression systems for production of recombinant proteins Bacteria Yeast Insect cells Mammalian cells