E-Learning Course: Recombinant DNA Biology Course Code: MSLSC2003C04 Course Coordinator: Dr. Tara Kashav Topic: Protein expression (part 2)

Note: All the material is compiled/modified from textbooks/freely available e-resources just meant for learning purpose of students Baculovirus/insect system

• Baculoviruses are insect viruses with ds DNA genomes • They infect their insect larval hosts – Acting as parasites – Killing the larvae & turning them into factories for virus production • Late in infection process – Viruses produce large amounts of two proteins – p10 and polyhedrin – NOT needed for virus production in cultured insect cells • The for both of these proteins can be used for – Expression of of a heterologous protein (Gene of Interest) Baculovirus Multicapsid nucleopolyhedrovirus • Rod shaped DNA virus that replicate in nuclei of insect cells, natural and specific insect pathogens • Double stranded circular genome 80-180 kbp Occlusion bodies • Protein lattices • They protect insect viruses (infectious particles) after release into environment • Keep long-term infectivity of tissues after the host death • At the end of infection, cell dies, occlusion body persists • When a infected larvae dies on leaf, occulsion bodies are deposited on it and can stay infectious until another insect arrives to eat the leaf and ingest the protected virions Baculovirus infection cycle

• pH 10-11 dissolves Obs • Viral genome reaches columnar cells nucleus • Viral occurs • New progeny virus assembled and released in OBs

Adapted from chambers et al 2018 Baculovirus/insect system

• Recombinant virus is used to infect insect cells or larvae – Heterologous protein is produced at very high levels – ~25% of total protein present at end of infection cycle • Baculovirus most often used for protein expression – Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) – Large genome (134 kbp), too large for direct cloning – Virus purification is also cumbersome Modifications in baculovirus/insect system

• These problems have been solved by the creation of bacmids – large circular DNAs – Include entire baculovirus genome – Along with sequences that allow replication of bacmid in E. coli – A shuttle vector that can be propagated in both E. coli and insect cells ex; sf9, sf21 Cloning in baculovirus/insect system

• Gene of interest is first cloned into a smaller plasmid – Between two sites (att) – Recognized by a site-specific recombinase • Introduced into baculovirus vector (larger plasmid) – By site-specific recombination, in vivo • Generates a circular DNA product – Used to infect the cells of an insect larva

Recombinant bacmid Recombinant bacmid

• Recombinant bacmid is then – Isolated – Transfected into insect cells – The gene of interest (GoI) is expressed during the infection cycle – GoI is downstream of a promoter that normally expresses a baculovirus coat protein at very high levels – Recovery of the protein once the infection cycle is finished • Wide range of bacmid systems is available commercially • Transfection – DNA used for transformation includes viral sequences – Leads to viral replication Recombinant Baculovirus vector expressing a protein that produces red color Advantages of Baculovirus/insect system

• Heterologous protein is produced at very high levels • ~25% of total protein present at end of infection cycle • High yields may lead to formation of inclusion bodies • Provides more features characteristic of mammalian cells – Post-translational modifications • Successful for phage display of large, complex disulfide- containing proteins Limitations of Baculovirus/insect cells

• Not successful with all proteins • Higher costs • Longer duration before you get protein (usually 2 weeks) Remember: Factors affecting gene expression • Facilities of laboratory and local expertise • Type of protein to be expressed • Whether the protein is toxic? • Whether carbohydrate or other modification required? • Requirement of large yield of protein • How to purify protein? • Cost of protein production • Regulatory and safety considerations. • Downstream applications of purified protein Mammalian Cell Culture

• It is the core for generating/biomanufacturing : – Therapeutic proteins – Viral vaccines – Functional tissues analogues for regenerative medicine Different vector systems

• To transfer the gene of interest into mammalian cells: – Plasmid based expression vectors – Adenovirus vectors – Vaccinia vectors – Retroviral vector – Baculovirus Plasmid based expression vectors

• The gene of interest is cloned under virus promoter in an – Expression is controlled by a virus promoter ex, SV40 or cytomegalovirus (CMV) – One non-viral promoter ex, elongation factor (EF)-1 as it appears to be strong than some viral promoters Adenovirus and Retroviral vectors

• Most convenient way to introduce cloned genes into a mammalian cell is with viruses – Natural capacity to insert DNA/RNA into a cell – At times into the cellular chromosome • Variety of engineered mammalian viruses are available as vectors – Human adenoviruses and retroviruses Adenovirus vectors

• Adenovirus is medium sized, non-enveloped icosahedral virus • Extensively studied virus • Composed of – a nucleocapsid – ds linear DNA genome- can be used as a cloning vector • For heterologous gene expression – Early regions E1 and E3 were deleted – This removes virus replication capacity – Host cell machinery used for virus replication • An expression cassette was set in place of the deleted E1 region • In this cassette – Recombinant gene placed under additional major late promoter as of cytomegalovirus • Ex of vector: pAdBM5 Vector map

Adapted from https://www.addgene.org/guides/adenovirus/ Requirements

• Suitable cell line – HeLa, HEK293T, U2OS, A549, HT1080, CAD, NIH 3T3, Human embryonic kidney 293 cells, MCF-7 etc. • Appropriate vectors – Act as a vehicle to transport gene of interest into cell lines – Ex: Plasmid based expression vectors, Adenovirus vectors, vaccinia vectors, retroviral vectors, Baculovirus Cell culture basics

Co2 Incubator Cell transfer/handling inside a laminar air flow

Check below link for cell culture basics: https://www.youtube.com/watch?v=CMRKKl9XSDU Process for transfer of Gene

• Process of transfer of gene is called Gene delivery • Implications: – gene therapy – genetic modification of crops Gene delivery • Process of introducing foreign DNA into host cells • Different methods of gene delivery developed for a various types of cells from bacteria to mammals – Non-viral methods – Viral methods Non-viral methods

• Physical methods – Electroporation – Microinjection – Gene gun • Chemical methods – Lipofection – Polymeric gene carriers Electroporation

• Under strong electrical pulse applied across a cell or tissue – Structures of cell or tissue would rearranged to cause permeabilization of cell membrane • Named in early 1980’s “electroporation” Microinjection

• Using a very fine needle to insert substances at a microscopic or single living cell – Needle ~ 0.5 to 5 μm in diameter penetrates the cell membrane and/or the nuclear envelope – Desired contents are injected into desired sub-cellular compartment & needle is removed – Performed under a specialized optical microscope setup called a micromanipulator Microinjection-transgenetics

• Simple transgenic organisms created by – Injecting genes into testicle of a nematode during meiosis of male gamete formation – All of the nematode's gametes will carry a foreign gene as the result of a single injection • Cloning of organisms Gene Gun

• Also termed – Ballistic DNA delivery/DNA-coated particle bombardment • First used for gene transfer to plants in 1987 (Al-Dosari and Gao, 2009; Lin et al., 2000) • Principle – Delivery of DNA-coated heavy metal particles by crossing them from target tissue at a certain speed – Particles achieve sufficient speed due to a pressurized inert gas (generally helium) – Gold, tungsten or silver microparticles were used as the gene carrier Gene Gun Advantages of Gene Gun

• Does not use toxic chemicals or complex biological systems • Delivery is achieved without the need for a receptor • DNA fragments of various sizes, including large ones, are transported • No need to introduce foreign DNA or protein • High reproducibility • Production of heavy metal particles is easy • Limitations: Gene expression is short-term and low Lipofection

• Cationic lipids forming micellar structures called liposomes are complexed with foreign DNA to create lipoplexes • The structures fuse with the cell membrane – Sometimes after interactions with surface proteoglycans. • DNA imported into nucleus might result in gene expression • Risk: DNA might be degraded within the lysosome Lipofection Receptor-mediated internalisation of ligand-labelled polyplexes

• DNA can be complexed with cationic polymer – Poly-ethylene-imine (pEI) – Conjugated with ligands specific to a target cell type - polyplex • Polyplexes can be used to – Target gene expression by docking with cognate receptor

Virus mediated gene delivery

• Virus mediated gene delivery utilizes – Ability of a virus to inject its DNA inside a host cell • A gene intended for delivery is packaged into – A replication-deficient viral particle • Viruses used to date include – Retrovirus, adenovirus, adeno-associated virus & herpes simplex virus Mode of Gene Delivery

• Allows integration of gene to be transferred into genome of the target cells – e.g.: retroviral and lentiviral vectors • Transfer transiently the new gene function to target cells – e.g.: adenoviral vectors, AAV vectors in which transgene will not be integrated Drawbacks

• Viruses can only deliver very small pieces of DNA into cells • Labor intensive • Risks of random insertion sites • Cytopathic effects and mutagenesis Application of mammalian expression system • First approved was tissue plasminogen activator (tPA) – Produced in 1987 by Genentech Inc. • 2006 to 2010: 58 biopharmaceutical products approved – 32 were produced in mammalian cells – 17 were produced in E. coli – Four in yeast – Three in transgenic animals – Two in insect cultures Examples of Successful high level production of following • Monoclonal antibody • Urokinase • Follicle stimulating hormone Other applications of mammalian expression system

• Novel mammalian cell lines expressing reporter genes – Used for detection of environmental chemicals – By activating endogenous aryl hydrocarbon receptors or estrogen receptors • Hemophilia B – Genetic disease of coagulation system – Affects one in 30,000 males worldwide – Recombinant human Factor IX (rhFIX) used for treatment – Stable, high-level of rhFIX produced in human hepatic cell lines Advantages

• proteins can be expressed either transiently – Viral DNA is maintained separately from host cell genome and eventually degraded • Permanently – Viral DNA is integrated into the host cell genome • With the correct choice of host cell – Proper post-translational modification of protein to its active form can be ensured Limitations

• Growth of mammalian cells in tissue culture is very expensive • This technology is generally used to – Test the function of a protein in vivo rather than to produce a protein in large amounts – Potential contamination with animal viruses of mammalian cell expression have been bottlenecks for its use in large-scale industrial production Expression in Plants

• Many plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens • A Ti or tumour inducing plasmid is a circular plasmid • GoI cloned into T-DNA, a stretch of DNA flanked by a 25-bp direct repeat sequence at either end, and which can integrate into plant genome • T-DNA also contains the selectable marker • Agrobacterium provides a mechanism for – Transformation – Integration into the plant genome – Promoters for its vir genes used for cloned genes Expression in Plants

• Some plant viruses may be used as vectors since Agrobacterium does not work for all plants. • Examples of plant virus used are the tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus. • The protein may be expressed as a fusion to coat protein of virus and is displayed on surface of assembled viral particles, or as an unfused protein that accumulates within plant. Schematic for expression in plants Particularly difficult cases

• where the protein of interest is toxic or prone to form inclusion bodies • the use of a cell-free protein biosynthesis system may be beneficial In Vitro Translation

• Important tool for molecular biologists • The in vitro synthesis of proteins in cell-free extracts • Applications – Rapid identification of gene products (e.g., proteomics) – Localization of mutations through synthesis of truncated gene products – Protein folding studies – Incorporation of modified or unnatural amino acids for functional studies In Vitro Translation

• The use of in vitro translation systems have advantages over in vivo gene expression if – the over-expressed product is toxic to the host cell – when the product is insoluble or forms inclusion bodies – when the protein undergoes rapid proteolytic degradation by intracellular proteases in vitro Translation Procedure In Vitro Translation

• In principle, it should be possible to prepare a cell-free extract for in vitro translation of mRNAs from any type of cells

• In practice, only a few cell-free systems have been developed for in vitro protein synthesis – These systems are derived from cells engaged in a high rate of protein synthesis. Type of cell-free translation systems

• Rabbit Reticulocyte Lysate • Wheat Germ Extract • E. coli Cell-Free System Rabbit Reticulocyte Lysate

• Highly efficient in vitro eukaryotic protein synthesis system – used for translation of exogenous • Reticulocytes are highly specialized immature red blood cells lost their nuclei – Primarily responsible for the synthesis of hemoglobin – contain adequate mRNA & complete translation machinery for extensive globin synthesis – More than 90% of the protein made in the reticulocyte • The endogenous globin mRNA eliminated by – incubation with Ca2+-dependent micrococcal nuclease – Later inactivated by chelation of the Ca2+ by EGTA Wheat Germ Extract

• Convenient alternative to rabbit reticulocyte lysate • Has low background incorporation due to its low level of endogenous mRNA • Wheat germ lysate efficiently translates exogenous RNA from a variety of different organisms – Viruses, yeast, higher plants and mammals • Recommended for translation of RNA which are inhibitory to the rabbit reticulocyte lysate • Small fragments of double-stranded RNA or oxidized thiols E. coli Cell-Free System

• Relatively simple translational apparatus • E. coli cell-free systems consist of a crude extract that is rich in endogenous mRNA – Extract is incubated during preparation so that endogenous mRNA is translated & subsequently degraded – If not, it is unsuitable for translation of RNA as exogenous RNA is rapidly degraded by endogenous nucleases – Some viral mRNAs (TMV) translate efficiently as they are resistant to nuclease activity • E.coli extracts are ideal for coupled transcription:translation from DNA templates. "Linked" & "Coupled" Transcription : Translation Systems • In standard translation reactions, purified RNA is used as a template for translation • "Linked" & "coupled" systems use DNA as a template • RNA is transcribed from the DNA and subsequently translated without any purification • Such systems typically combine – a prokaryotic phage RNA polymerase – Promoter (T7, T3, or SP6) – Eukaryotic or prokaryotic extracts • DNA templates may be – Cloned into plasmid vectors – Generated by PCR Reading

• Chamber’s et al 2018, review article • Lehninger’s book • Primrose, S. B., & Twyman, R. (2009). Principles of gene manipulation and genomics. Wiley. com. • Brown, T. (2010). Gene cloning and DNA analysis: an introduction. John Wiley & Sons. • PMC article entitled “Gene Expression in Mammalian Cells and its Applications” by Kishwar Hayat Khan • https://www.addgene.org/guides/adenovirus/