; LOSCHMIDT , LABORATORIES Protein expression & purification technologies Manufacturing of recombinant proteins, protein complexes and virus-based vehicles for biotechnology and basic research Dr. Martin Marek Loschmidt Laboratories Faculty of Science, MUNI Kamenice 5, bid. A13, room 332 martin.marek@recetox.muni.cz What will we talk about • Introduction to recombinant protein production • definition, goals and applications • Protein expression in microbial cells • Escherichia coli, yeasts • Expression and multi-expression in eukaryotic cells • Mammalian and insect cells, baculovirus expression • New emerging expression tools • CRISPR/Cas9 technology, cell-free protein expression • Protein purification, characterization and storage • Chromatography separation & QC check Why to purify a protein? • To study its function • To analyze its physical properties • To determine its sequence • To determine its three-dimensional structure • For industrial or therapeutic applications 3 Applications of protein technologies 4 LOSCHMIDT LABORATORIES The key role of protein production in pharma industry 10 ZÜ SVG IVYGDQYRQLCCSSPKFGDF iS I G I VYGDQYRRVCCSSPK FGD 3VGIVYGDQYRRLCCSSPKFGD 3VGIVYSSEIHRLSDLSPKYGNF .01 HN Identify target (Genomics, biological assays) Isolate protein involved in disease (Molecular cloning, recombinant expression, biochemical and structural analyses) 1038 ,unAf o- ir-zr, - a Att _l g,5 p[ Find a drug effective against disease protein (virtual screening, HTS and library screening etc.) Preclinical testing (Animal studies, scale-up, formulation) Human clinical trials Protein production is a significant bottleneck in early phase drug discovery FDA approval 5 OtNijstalJ |jr íniKtľ í Recombinant protein production workflow • Molecular cloning • Protein expression • Protein purification • Protein characterization • (Protein structure determination) 6 LOSCHMIDT LABORATORIES Cellular protein production: selection of a host Escherichia coli • Insect cells (Baculovirus expression system) Yeasts (Pichia, Kluyveromyces) • Mammalian cells (HEK293) SPEED COST TYPICAL YIELD POST- TRANSLATION MODIFICATION LOW MAMMALIAN BACTERIA BACTERIA BEVS/INSECT CELL YEAST YEAST BEVS/INSECT CELL Si A MAMMALIAN BEVS/INSCaaU BACTERIA YEAST BEVS/INSECT CELL HIGH BACTERIA MAMMALIAN &j9 YFAST MAMMALIAN LOSCHMIDT LABORATORIES Bacterial expression system Concepts Methods Applications 8 Recombinant protein expression in Escherichia coli ADVANTAGES • Inexpensive setup and running costs • High recombinant protein production levels • Short timeline from cloning to protein recovery (1 week) • Limited technical knowledge required for culturing • Scalability from small (2 ml_) to very large culture (>10,000 L) volumes DRAWBACKS • Inability to perform post-translation modifications (PTMs) • Limited formation of disulphide bond Workflow of protein expression in E. coli Transform plasm id into E. coli Grow cells and induce expression with IPTG_ Collect bacterial pellet by centrifugation Recombinant plasmid Protein expression in E. coli: a plasmid backbone • pET • pGEX Thioredoxin (Thx) Maltose-binding protein (MBP) Glutathione S-transferase (GST) Small ubiquitin-like modifier (SUMO) LOSCHMIDT LABORATORIES Co-expression of protein complexes in E. coli The functional units within cells are often macromolecular complexes rather than single species. Production of these complexes as assembled homogenous samples is a prerequisite for their biophysical and structural characterization and hence an understanding of their function in molecular terms. Co-expression in Escherichia coli can decipher the subunit composition, assembly, and production of whole protein complexes. Cut and Paste Gene A Bglll Spel pnEA-His ^—IQRI C01E1I—JAmpiciHn Bglll Spel Xbaf pnCS ^3-»KZM" GeneB r Bglll Spel pnEA-His Xbal OR! ColEU—Ampicilinl Example of the concatenation of two vectors of the pET-MCN vector series: pnEA-His (vector encoding an N-terminal poly-histidine tag in front of protein A) and pnCS (vector expressing the native protein B). The pnEA-His (acceptor vector) is linearized by removing with the restriction enzymes Bglll and Spel part of its promoter (T7 promoter and lacO). The full promoter of the pnCS (donor vector) is cut out with the restriction enzymes Bglll and Xbal. After ligation of the pnCS promoter with the linearized pnEA-His vector, a new vector is obtained based on the backbone of the pnEA-His vector and whose promoter controls the genes of proteins A and B. LOSCHMIDT LABORATORIES Wide range of E. coli strains Strain Features BL21 (DE3) Most common host strain, enables high-level recombinant protein expression BL21 (DE3) pLysS Enables high-level expression and suppression of T7 RNA Polymerase basal level expression BL21-CodonPlus Improved expression of genes with codons rarely used in bacteria Rosetta Improved expression of genes with codons rarely used in bacteria Arctic Express Contains a plasmid encoding chaperonins to aid in folding and allow expression at low temperature (12°C) Shuffle T7 Express Expresses DsbC to enable cytoplasmic disulphide bond formation 12 Expression in E. coli: tips and tricks • Growth temperature: Typically 37°C, but lowering (25°C or 15°C) improves folding/solubility • Expression with a fusion tag: polyhistidenes (6xHis, 10xHis or 12xHis) Thioredoxin (Thx) Maltose-binding protein (MBP) Glutathione S-transferase (GST) Small ubiquitin-like modifier (SUMO) • Co-expression: Chaperonins (folding) and foldases (disulphide isomerase, DsbC) Co-expression with interaction partners (co-solubility effects) • Media: LB Broth, 2xLB Broth (simple and cheap), Terrific Broth (TB), 2YT • Antibiotics: Selection of recombinant clones and prevention of contamination Concentration of antibiotics in large-scale expression is decreased • Codon optimization: Modifying codons in a gene sequence to match the codon usage bias of the host cell used for expression 13 LOSCHMIDT LABORATORIES Expression in yeasts Concepts Methods Applications 14 LOSCHMIDT LABORATORIES Expression in yeasts The yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris, constitute another microbial systems in heterologous protein expression. GaUp produced Irom ihe GALA promoter Figure 8.S. Galactose inducible gene expression in yeas!. The expression of genes from multicopy vectors under the control of the GAL] promoter (P- f-* 29 Selected vaccines produced by BEVS Application Product name Company Stage References For human use Cervical cancer CERVARLX® GSK Approved Prostate cancer PROVENGE® J e lj ::.: e co Approved !n em;. Protein Science: Approved A/HrMl Virus-like particle Novavan Phase [ (NCT&1S9671S) For vet erinary use Procrine cnicoviriis 2 (PCVz) Porcihs® PCV Merck Approved PCto GrcoFLEX® Boehringer Ingelhe im Approved [35] Swine fever Porcihs Pesti® Merck Approved LOSCHMIDT LABORATORIES 30 , LOSCHMIDT ; LABORATORIES Types of COVID-19 vaccines C0VID19 Vaccine platforms designed to train our immune system VACCINE TRACKER 1 T TYPE E OF COMPONENT VIRAL VACCINES _I_ T Protein Subufiil Cwitatre JKÜWl and purified viral prcrU'in> Virus-like particles ('-'LPl DNA-biiscd RNA-based Nop-Re p li eating Virtl Vettor Caitaiis viral proteins thjrňňiŕriiCJhtttíUtCurfl pf the 'rtruE. (kit no Ľ.:.ľLi ľ', b'ir.il rji-i.i In in,Hi"..i liUCh 35 rnRNAf iwJi-trh provider live insuüctlQris few making viral proteins ■ Cöftliim viral gPftrtlC nui(f rcjil packaged injide ancrchef harmlesi virus trial «n r*pt topy Üself 9 Ö 1 Ty PES OF WHOLE VIRUS VACCINEN ._I_ Replicating Viral VedtOr Contairii viral genetic rnaterral packaged inside anoi-hŕr h.umlMi virui \t\H an copy rtseli 1 Inactivated JL Live.-Attenuated ■ Gmiaifli tßpi« öl I hi h'ir.., Ihül I'.i.T brtfi hilled ÍBií1Ct"?tíd I Contains ropie* of the virus that have been weskrppj iaLtenudiKfl SARS-CoV-2 is trie1 virus, that causes COVlD-1^. Thp Epik ŕ protein on the surface of SAR5-COV-2. is in example a1 an antigen. Vsiclnflsaretlie best way to train our immune System tú- retogniie viruses. Or pieces. OÍ viruses, called antigens. Our immune Extern creates antttwdies and other defenses to puotett us. When a vaccinated person is exposed to SAflS-CoV*2, their immijpe system will recognize the viral antigens and spring into action to keep them healthy. There are many different types erf vaccines, as. shown above. 31 LOSCHMIDT LABORATORIES Baculovirus-mediated production of gene therapy vectors Baculovirus-mediated production of adeno-associated viral (AAV) vectors as gene therapy vehicles a. Standard production [Čap] [Transgene Baculovirus Infection Purification rMV production in insect cells (sf9) AAV virus particles b. One Bac Baculovirus infection #5#5# Purification I cells expressing essential AAV genes rAAV production AAV virus particles ITR r.p5 r-Pl9 r»p40 AAV Poly A 1 TR wtAAV -r~ cap Sj 4.7 Kbp ITR rAAV Hp romoter Poly A 1 Thera peutic gene ITR a In 2002, Prof. Robert Kotin and colleagues at the US National Heart, Lung, and Blood Institute first demonstrated its suitability for AAV manufacturing They infected Sf9 cell lines — derived from the fall armyworm — with three different baculoviruses: two containing essential genes for AAV particle production (rep and cap), and one containing the transgene sequence intended for delivery In this manufacturing process, the baculoviruses play a dual role, functioning as the 'helper' virus normally required for replication, as well as the vehicle for AAV genetic material In their initial demonstration, Kotin's team achieved levels of productivity comparable with existing AAV manufacturing approaches — on the order of 50,000 functional viral particles per cell LOSCHMIDT LABORATORIES Development of AAV-based gene therapy products In 2017, FDA-approval of the first gene therapy product targeting a disease caused by mutations in a single gene This product, LUXTURNA™ (voretigene neparvovec-rzyl; Spark Therapeutics, Inc., Philadelphia, PA), delivers a normal copy of the RPE65 gene to retinal cells for the treatment of RPE65 mutation-associated retinal dystrophy, a blinding disease Many additional gene therapy programs targeting both inherited retinal diseases and other ocular diseases are in development LUXTURNA" voretigene neparvovec-rzyl About LUXTURNA Take Your First Step Patient Support LUXTURNA is the first FDA-approv gene therapy for a genetic disease LOSCHMIDT LABORATORIES World's most efficient large-scale AAV manufacturing VIROVEK -ft HOME K COMPANY - : AAV PRODUCTION v OTHER SERVICES v- Virovek has developed a patented BAC-to-AAV technology that utilizes the baculovirus expression system to produce AAV vectors in insect cells under serum-free condition The capability to generate over 3e+16vg of AAV vectors with a single production run, which is unmatched by any other AAV production system BAC-TO-AAV TECHNOLOGY FOR LARGE SCALE AAV PRODUCTION Applications of BEVS (a) (Glyco-)proteins, subunits and (e)VLPs i Surface display, antigen carrier (b) AAV triple infection (rep, cap, GO I) Viral vectors BVs Gene delivery Overview of the various applications of the baculovirus expression system. Nowadays, baculovirus-insect expression system has been relatively mature in many labs for protein production and is becoming a leading method to produce high quality proteins among the eukaryotic expression systems The use of BEVS to express the protein of interest requires two steps. In the first step, the insect cells are cultured to the desired concentration. In the second step, the baculovirus is applied to infect the insect cells. The virus that infects host cells will dominate the gene expression mechanism in the host cell and trigger the production process of the target protein Yet, several mature expressing vectors have been developed, including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that is the most extensively used The ability to produce biologically active mammalian proteins makes the BEVS a powerful tool over yeast and bacterial expression systems Applications of baculovirus technologies Advantages and drawbacks of baculovirus expression ADVANTAGES • Recombinant protein has complete biological function, such as protein correct folding and disulfide bond • Post-translation modification (glycosylation etc.) • High level of expression, up to 50% of the total protein amount • Accommodate large insert protein • Simultaneously express multiple genes (multi-expression) • In general, it is safer to use than mammalian virus, since it has limited host range and does not infect vertebrates • The drawback is that exogenous protein expression is under control of the very late viral promoter, where the cells begin to die due to viral infection • Insect expression system is normally used for production of membrane proteins, although the glycosylations may be different from those found in vertebrates LOSCHMIDT LABORATORIES Virus-free expression in insect cells duration in weeks 1 0.5-1 2-12 3-8 -- /Oq Cloning of the ^ \Q expression vector y I Transient transfection of insect cells Transient protein production -m. Selection of stable polyclonal cell lines 3 npr Stable protein production using a polyclonal cell line ^Single cell cloning and screening for high producers 1 2 3 4 5 8 7 £ $ 1Ů 11 13 oooooooooooo soooooooooooo < oooooooooooo i' oooooooooooo Eoooooooooooo ►oooooooooooo oooooooooooo Hoooooooooooo Expansion and scale up -optimization of process conditions Stable protein production using a monoclonal cell line Overview of the general procedure to produce stably transformed D. melanogaster S2 cell lines for recombinant protein expression Protein expression can be initiated at different points, starting with transient expression immediately after transfection followed by stable expression in a polyclonal cell line and finally the selection of a highly productive monoclonal cell line 38 3, LOSCHMIDT LABORATORIES The different plasmid sets to generate stable S2 cell lines c Most likely integration of multiple copies X Integration of single fragments. 3 a) Two separate plasmids T ■ fm 1:11 VŮCQDC ^\>) One plasmid witr^ separte promoters c) One mulllclstronic plasmid Plasmid Willi trans poson t*t\>v Iii«™) e) RHCE I \ Plasmid i coVjivng .http* ( _ u&unfl J /"\ plasmid backbone Key loariL-nning o,g. origin aFraplirjilion rind nofcrion i^ttaflBfwiircprignrliari in bacteria) constitutive promoler selection cgs-sette ' (cmraining .nnlibtalk rnsASbinc? mnrkrMl constitutive or inducible promoter iu A.Sar P.Hi: expression cassette (TOnl^mlng eg. tiW-3K wqu*«M, pgrisl poplK^o-. g*n« or inlgiiosL ivvsri poly A ItflrijIJ 4 internal ribosome enlry site (IRES) / 2A-like sequence (T2A) cassette encoding a transposese (i.e. P lrtrt$pp*J$d/MJio* Iramapcsatn) transposase recognition sites. ■.i...-. P i..\:i:i\'.ti\ turinrrf :epe;tta I'AAiriDu; nverf&d n^ji-^il^-! cassette encoding a reporter protein AM Coro- DNA Mew DNA N on-homo logo us end joining (NHEJ) H ü I no I ü g v direcled repair (HDH) LOSCHMIDT LABORATORIES Cell-Free Protein Synthesis (CFPS) CELL-FREE PROTEIN SYNTHESIS IN VIVO PROTEIN SYNTHESIS Transformation and Expression • Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression • The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest 49 LOSCHMIDT LABORATORIES Cell-free platforms and their applications (A) Web of the applications enabled by low adoption cell-free platforms. Connections shown are based on applications that have been published or that have been proposed in publications. (B) Cumulative number of peer-reviewed publications over the last 60 years for cell-free platforms. Preparation of cell-free extract and set-up of CFPS reactions Growth Harvest 1 1 1 - - Pre Lysis Lysis DNA Template j Cofactors and Substrates TP" Energy and Salts Post-Lysis Cell-Free Protein Processing Synthesis Reaction General workflow for preparation of cell-free extract and set up of CFPS reactions. A visualization from cell growth to the CFPS reaction is depicted above for a new user, highlighting the main steps involved. 51 LOSCHMIDT LABORATORIES Comparison of CFPS platforms BATCH CONTINUOUS EXCHANGE Substrate-Rich Feed Solution r Dialysis Membrane V CONTINUOUS FLOW Substrate-Rich ' Feed Solution o o 0 ö 0 Ultrafiltration Membrane ofe................. '"7rQv°"o'"^"o' \ Product-Rich Output Q Template DNA Substrates )Q Active Ribosome Byproducts [J GFP Reporter A. Batch reactions contain all the necessary reactants within a single reaction vessel. B. Continuous exchange formats utilize a dialysis membrane that allows reactants to move into the reaction and byproducts to move out, while the protein of interest remains in the reaction compartment. C. Continuous flow formats allow a feed solution to be continuously pumped into the reaction chamber while the protein of interest and other reaction byproducts are filtered out of the reaction. , LOSCHMIDT ; LABORATORIES A GUIDE TO THE TWENTY COMMON AMINO ACIDS AMINO ACIDS ARE THE BUILDING BLOCKS OF PROTEINS IN LIVING ORGANISMS. THERE ARE OVER 500 AMINO ACIDS FOUND IN NATURE - HOWEVER, THE HUMAN GENETIC CODE ONLY DIRECTLY ENCODES 20. 'ESSENTIAL'AMINO ACIDS MUST BE OBTAINED FROM THE DIET, WHILST NON-ESSENTIAL AMINO ACIDS CAN BE SYNTHESISED IN THE BODY. Chart Hey: ALIPHATIC AROMATIC ACIDIC BASIC HYDROXYLIC SULFUR-CONTAINING AMI DIC O NON-ESSENTIAL I I ESSENTIAL NAME O three tetter cade UNA cottons ALANNE0 Ala tit I, lit!, litA, ütli GLYCINE G Gly Mil. ütit.ütiA, ütiS isoleugineO lie Al I.Alt, A1A NH \ \ OH I LEUCINE O Leu t II. tit, tlA. t 10 I IA I It. PROLINE O Pro CCl. ttt, Ltfi, ttü I OH I NH, \ / _ *<* VALINE O Va! ÜI l.tjlt. O'VUt PHENYLALANINE Phe TTT.TTf TRYPTOPHAN 0 Trp Ter, TYROSINE 7yr ASPART1CACIDQ GLUTAMIC ACID O TAT, TAC c;atp gat r~*A,r.AT, ARGININE O Arg ca, rcr, rr.A, rer.. .'«"„',. A;",r. / 1 € ii ^ HN-\ \ ° \ OH I HISTIDINE O \ o \ OH LYSINE O AAA, AAC SERINE 0 TfT, T This chart only shows those amino acids for which the human genetic code directly codes for. Selenocysteine is often referred to as the 21st amino acid, but is encoded in a special manner. In some cases, distinguishing between asparagine/aspartic acid and glutamine/glutamic acid is difficult, In these cases, the codes asx (B) and glx (Z) are respectively used. ©COMPOUNDINTEREST2014-WWW.COMPOUNDCHEM.COM | Twitter: @compoundchem | Facebook: www.facebook.com/compoundchem Shared under a Creative Commons Attribution-Noncommercial NoDcrivativcs licence. BY HC HQ LOSCHMIDT LABORATORIES CFPS reaction incorporating nonstandard amino acids Extract ^r4Sv^'" Preparation^, o-tRNA mRNA , Assess the impact of RF1 deletion on cell-free protein synthesis (CFPS) vs. o .............' Ribosome prfA strain: AprfA strain: competition no RF1 with RF1 p-acetyl-L-phenylalanine Scheme of cell-free protein synthesis reaction incorporating nonstandard amino acid to investigate the effect of RF1 Cell extracts containing transcription and translation machinery are prepared from rEc.E13 or rEc.E13.AprfA strains Plasmid DNA template of sfGFP containing single or multiple amber codon sites, orthogonal tRNA/aaRS NSAA, T7 RNA polymerase, and other cofactors are added as necessary to activate the cell-free protein synthesis (CFPS) reaction LOSCHMIDT LABORATORIES CFPS from extracts of a genomically recoded organism nature =/\LV COMMUNICATIONS V 3\ ^ ^^C-Pss Extract pEVOL aaRS Cell-free reagents C321.AA.759 + pEVOL Plasmid Orthogonal tRNA Orthogonal aaRS 05 Rey W. Martin, Benjamin J. Des Soye, Vong-Chan Kwon, Jennifer Kay, Roderick G. Davis, Paul M. Thomas, Natalia I. Majewska, Cindy X. Chen, Ryan D. Marcum, Mary Grace Weiss, Ashleigh E. Stoddart, Miriam Am Irani, Arnaz K. Ranji Charna, Jaymin R. Patel, Farren J. Isaacs, Neil L. Kelleher, Seok Hoon Hong & Michael C. JewettH Nature Communications9, Article number: 1203 (201&) Download Citation i 2802 Accesses 25 Citations 86 Altmetric Metrics » CFPS UAG = ncAA <*) Schematic of the production and utilization of crude extract from genomically recoded organisms with plasmid overexpression of orthogonal translation components for cell-free protein synthesis CFPS reactions are supplemented with the necessary substrates (e.g., amino acids, NTPs, etc.) required for in vitro transcription and translation as well as purified orthogonal translation system (OTS) components to help increase the ncAA incorporation efficiency • aaRS, aminoacyl tRNA synthetase; ncAA, non-canonical amino acid; T7P, T7 RNA polymerase; UAG, amber codon LOSCHMIDT LABORATORIES New course in English available! Bi9690en Synthetic Biology Dr. Karel Říha karel.rihalKiceitec.rriuni.cz Dr. Martin Marek ma ri nt. m a rek(SJ recetojc.muni.cz Synthetic biology is a new scientific discipline that builds on advances in molecular biology, genetic and protein engineering, systems biology and bio inform a tics, to re-des'gr ex'sihg biologka sys:eir s 'or applicat'ons 'r biotechnology and medicine. In this course, students will grasp concepts in synthetic biology, become familiar with basic methodological approaches and learn about its applications in science and technology, Selected topics: • Basic concepts of engineering in biology • From genetic engineering to synthetic genomes • Protein engineering and design • Expanding building bricks of life • Fro m protei ns to na no ma ch i n es • Metabolic engineering, artificial organelles • Future trends in synthetic biology and ethical issues LOSCHMIDT LABORATORIES https://loschmidt.chemi.muni.cz/peg/ II u II i LOSCHMIDT A TUESDAYS A START 21/09/2021 A Room 333, Building Bll U University Campus, Brno 56 Molecular Biotechnology Practicals Kdy: Úterý 11.10. 2022 od 10:00 od 14:00 Kde: D36-308 57 LOSCHMIDT LABORATORIES Questions 58 LOSCHMIDT LABORATORIES Protein purification methods Concepts Methods Applications 59 3, LOSCHMIDT LABORATORIES Recombinant protein purification: step by step The aim of a purification procedure is to obtain a highly pure and stable protein at an appropriate concentration in a buffer compatible with the intended application. Recombinant protein expression o Clone \^ Transform Strip Wash Buffer Buffer Elute Wash — Chromatography steps Affinity chromatography Ion-exchange chromatography Size-exclusion chromatography Hydrophobic interaction chromatography Reversed phase chromatography Lyse cell pellet (Sonication, high-pressure homogenizer, Microfluidizers) Pellet m Clarify cell lysate by high-speed centrifugation 60 Chromatography columns in protein purification Chromatography is the most powerful and commonly used means of purifying recombinant proteins. Each technique separates proteins based on different properties, so it is often advantageous to combine several types to maximise separation of the recombinant protein from host cell proteins. Technique Affinity Chromatography (AC) Ion Exchange Chromatography (IEX) Hydrophobic Interaction Chromatography (HIC) Size Exclusion Chromatography (SEC) Reverse Phase Chromatography (RPC) Stage Capture or Intermediate Capture or Intermediate Intermediate Polishing Description Based on a reversible interaction between the protein/affinity tag and a specific ligand Separates proteins based on their net surface charge Binding under high salt conditions, generally performed following an ammonium sulphate precipitation step Separates proteins based on their hydrodynamic volume (size) High-resolution chromatography based on weak hydrophobic interactions. Harsh conditions generally only suitable for purification of peptides 61 Affinity chromatography: fusion tags Fusion tags can improve protein expression, stability, resistance to proteolytic degradation and solubility. Fusion tag Function Size (kDa) Description Polyhistidine (e.g. 6xHis, 10xHis) Affinity 1-2 The most commonly used affinity tag, binds to metal ions Strep-tag II Affinity 1 High affinity for engineered streptavidin Thioredoxin (Trx) Solubility 12 Aids in refolding proteins that require a reducing environment Small Ubiquitin-like Modifier (SUMO) Solubility 12 Contains a native cleavage sequence enabling tag removal with SUMO protease Glutathione S-transferase (GST) Solubility, affinity 26 High affinity for glutathione, often needs to be removed due to large size Maltose Binding Protein (MBP) Solubility, affinity 41 Binds to maltose, often needs to be removed due to large size • Fusion tag orientation (N- or C-terminus) • Combinatorial fusion tags (Trx/GST/MBP with an affinity tag, e.g. 6xHis) LOSCHMIDT LABORATORIES Variety of proteases for fusion tag removal Human Rhinovirus (HRV 3C) PreScission protease Tobacco Etch Virus (TEVp) SUMOp Thrombin Protease recognition sequence _ Talon resin J L Talon Polyhistidine structure tag Recombinant protein 250 v 130-100" 70-55- 35-25- 15- 10- m ® ° -His-Thx-DAC1 -DAC1 63 LOSCHMIDT LABORATORIES Column chromatography instrumentation Chromatography steps Affinity chromatography Ion-exchange chromatography Size-exclusion chromatography Automated chromatography systems Protein characterization: an aggregation problem A key challenge in recombinant protein production is to maintain and store the target protein in a soluble and stable form. Protein aggregation can compromise protein function and thus it is necessary to overcome this challenge to generate functionally active protein. Detection Of protein aggregation Protein aggregation • Analytical size-exclusion chromatography (SEC) • Dynamic light scattering (DLS) • Analytical ultracentrifugation (AUC) Troubleshooting • Culture conditions (e.g. reducing temperature) • Buffer composition (ionic strenght, pH, reducing agents) • Presence co-factors (Acetyl-CoA, metal ions) • Fusion tags (Trx, MBP, SUMO) • Minimising sample handling • Avoiding time delays between purification steps • Performing purification steps at 4°C • Store purified proteins in -80°C LOSCHMIDT LABORATORIES Protein quality control (QC) analyses High purity and homogeneity of the protein sample are crucial for the downstream processes to be successful. Dynamic light scattering (DLS): To characterize the polydispersity of sample 60 n I 30- % Pd < 20 0-1E-1 1E+0 1E+1 1E+2 Identification of different oligomeric forms or aggregates, which are preventing crystallization 1E+0 1E+2 Radius (nm) Monodisperse Radius (nm) Polydis perse Differential scanning fluorimetry (DSF): analysis of protein stability To characterize the stability of the protein in different buffers and in the presence of different ligands, which stabilize the protein for crystallization Temperature {°C} Prometheus NT.48 (nanoDSF) LOSCHMIDT LABORATORIES Success stories 67 Development of expression and purification protocol for Schistosoma mansoni HDAC8: mini-scale tests LOSCHMIDT LABORATORIES 24-well deep plate for E. coii cultures 96-well deep plate for purification Tecan robot 50 mM KCl 10 mM Tris + + + + + + + + + + + + + + + 0.5% NP-40 - + - - + - - + - - + - + 5mM BME — - + - - + - - + - - + - - + kDa 130— 72-57-36— 28 — 17-I induction at ODeoo —— — ZZ — i 0.4 0.7 1.0 1.5 2.0 Semi-automated affinity purification smHDACS Induction at high O.D. results in higher yield Harvest cells 1 h post induction >8 , LOSCHMIDT ; LABORATORIES Large-scale production and crystallization of smHDAC8 600 500 ZD < E 400 Ü & .O O n < 300 H 200 100 Ion-exchange chromatography Peak smHDACS 10 15 20 30 40 Elution volume (ml) Moo heo -g h60 £ CT o U40 -20 I o Ü 50 60 B 120- >« 100 < E 80' i_ o < 4Q 20 A ■H Size-exclusion chromatography Peak smHDACS 0 20 40 60 80 100 120 140 160 Elution volume (ml) 140 >l 120 Ü CO ■J—- O O CO tu x> hHDACS smHDAC8 smHDAC8 69 LOSCHMIDT LABORATORIES Conclusions • The project design is ultimately determined by the end-use of the recombinant protein • The overall success of a project lies in an effective project design 70 LOSCHMIDT LABORATORIES Resources Blag Journalists CISION PR Newswire Sign Up News Products Contact Send a Release Search Q News in Focus Business S Money Science & Tech Lifestyle & Health Policy & Public Interest People & Culture Recombinant DNA Technology Market Worth $844.6 Billion by 2025: Grand View Research, Inc. GRAND VIEW RESEARCH 71 LOSCHMIDT LABORATORIES Questions 72 Dr. Martin Marek Loschmidt Laboratories Faculty of Science, MUNI Kamenice 5, bid. A13, room 332 martin. marek@recetox. muni, cz L LOSCHMIDT ; LABORATORIES LOSCHMIDT LABORATORIES Second Letter t C A 3 1 T SI"" TCT -i TCC TCA tcgj TAT 1 _ TAG J ^ TAA smp TAG Stop TQA Stop TGG Trp T c a g C ctgj CCT-) CCC cca ccgj ►Pro CAc}H,s CAA1 rin CAG J CGTT CGA f Arfl cggj T C a g a attt ATC \ He ATA j ATG Met acti ACC ACA acgj ^Thr AAT 1 . AAC J A9n mg>* AGTl -AGCJ &er T C A G GTT-i GTC Li GTA f™ gtgj GCTi GCC GCA gcgj GAT] . GAG] A*P GAA]Glu GAGJ ggtt GGA fGl¥ gggj T C A g 74 LOSCHMIDT LABORATORIES Baculovirus display system Baculovirus is a large DNA insect-infecting virus Baculovirus surface glycoprotein Gp64 is expressed early and late in the infection of an insect cell It is a 64 kDa protein which forms trimers and locates in the BV envelope with a polarized distribution As Gp64 is a transmembrane protein that exposes an outer domain, it can be used to display a selected protein on the BV surface A chimeric Gp64 can be constructed to contain the protein of interest allowing it to be incorporated in the BV structure upon infection of insect cells 75 LOSCHMIDT LABORATORIES The MultiBac: a factory for synthetic virus-like particles • A plasmid module comprising expression cassettes for the capsid-forming influenza H1N1 M1 protein (colored in grey) and a fluorescent protein marker, mCherry (colored in red), was introduced into the MultiBac baculoviral genome by Cre recombinase enzyme mediated plasmid fusion into the LoxP site (circle filled in red, gradient) • Co-expression of HA, NAand M1 yields synthetic influenza virus-like particles (VLPs) resembling live influenza virus 76 Baculovirus biology • Extracellular enveloped baculovirus virions can be found in two forms: OV (occluded virus) and BV (budded virus). The nucleocapsid is about 21 nm x 260 nm. • Circular dsDNA genome , 80-180 kb in length, encoding for 100 to 180 proteins. Caps id base ■""^ BACULOVIRUS INFECTION CYCLE • Attachment of the viral glycoproteins to host receptors mediates endocytosis of the virus into the host cell. • Fusion with the plasma membrane. • The DNA genome is released into the host nucleus. • Immediate early phase: host RNA polymerase transcribes viral genes involved in the regulation of the replication cascade, prevention of host responses and viral DNA synthesis. • Late phase: The virally encoded RNA polymerase expresses late genes. • Replication of the genome by rolling circle in nuclear viral factories. • Nucleocapsids are formed which can either bud out through the cellular membrane and disseminate the infection or be occluded for horizontal transmission. • Occlusion phase: the virus becomes occluded in the protein polyhedrin and the polyhedral envelope (calyx) is produced. • Lysis of the cell releases the occluded virus. LOSCHMIDT LABORATORIES The replication cycle of baculovirus Dissolution of OB in gut " ^ Fusion of ODV to midgut cells Secondary infection Budding Recombinant protein production VLP production BEVSjr^ Vaccines Gene therapy Tissue engineering Drug discovery Primary infection OB released upon cell lysis Baculovirus is a member of the insect-borne virus family and was originally used as a biopesticide with a large double-stranded circular DNA genome. The virus is enveloped, rod-shaped particles, ranging from 30 to 60 nm in diameter and from 250 to 300 nm in length. The natural hosts for these viruses are insects, and Autographa californica (AcMNPV ) is the most studied member of the family, which was developed into a recombinant baculovirus vector and is still under application today. AcMNPV and other baculoviruses are able to generate "polyhedra" or "occlusion bodies" in the infected cells of insects. In the late phase of infection, dozens of polyhedra are formed in the host cell nuclei, which accommodate progeny virions encased by a protective paracrystalline array comprised of virus-encoded protein-polyhedrin. LOSCHMIDT LABORATORIES A comparison of BEVS processes with low and high MOI primary infection of partial cell population Low MOI process virus release and secondary infection 0.1-0,01 0 cell lysis o 1 primary infection of the whole cell population High MOI process virus release, but no secondary infeclion E o if, c 24 h 96 h 24 h 96 h 79 How can baculoviruses express very late genes at such high levels? There is no evidence that the baculovirus RNA polymerase has an intrinsic ability for high level gene expression. In fact, it may not bind to the very late promoter region with high levels of tenacity as reflected in the fact that the footprint of the polymerase on genomic DNA has never been reported despite an extensive effort by at least one laboratory. It is likely that high levels of gene expression are influenced by several features of baculovirus biology. These include: i) the amplification of genes by DNA replication; ii) the shutoff of most late transcription, possibly by DNA binding proteins that coat the DNA and thereby make RNA polymerase available for very late transcription; iii) the efficiency of the late polymerase and VLF-1 in recognizing and initiating from very late promoter elements; iv) the efficiency of LEF-4 in capping the mRNA a possible role for LEF-2 and PK-1. As mentioned above, the 5' untranslated region of p10 mRNA appears to be capable of facilitating cap-independent translation, which may reduce the reliance of these transcripts on LEF-4 activity. Other factors that might enhance translation of very late expressed mRNAs have not been identified. 80 Baculovirus-mediated production of AAV vectors Gene transfer and gene therapy are powerful approaches for many biological research applications and promising avenues for the treatment of many genetic or cancer diseases. The most efficient gene transfer tools are currently derived from viruses. Among them, the recombinant adeno-associated viruses (AAVs) are vectors of choice for many fundamental and therapeutic applications. The increasing number of clinical trials involving AAVs demonstrates the need to implement production and purification processes to meet the quantitative and qualitative demands of regulatory agencies for the use of these vectors in clinical trials. In this context, the rise of production levels on an industrial scale appeared essential. The introduction, in 2002, of an AAV process using a baculovirus expression vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in expanding the AAV production in insect cells has been to switch the process to bioreactor systems, which are the ideal equipment for scaling up. We describe here a method for producing AAV vectors using the BEVS which can be easily used by research laboratories wishing to overcome the difficulties associated with the scaling up of production levels. The method provides sufficient quantities of AAV vectors to initiate preclinical projects in large animal models or for research projects where a single batch of vectors will consolidate the repeatability and reproducibility of in vitro and especially in vivo experimental approaches. 81 LOSCHMIDT LABORATORIES LEXSY - Eukaryotic protein expression in L tarentolae Most frequent an extensive / post-translatic the cloning ve suitable vecto grown in large precludes the removing prec boundaries of N- and/or C-te the bacterial s cell-free syste clones, strains Organisms and genes of interest In silico analysis - Domains - Post-transcriptlorial modifications - Secretion signals - Organelle targeting Gene cloning ■ Codom harmonization Purification lags * Solubility lags Gateway syslem (bo* 2) Homologous/Heteralogous systems e.g. Lstebmariia (box 1) Bacteria OOO Vra si P Baculovirus/lrtsect cells Cell -free systems O O Optimization - Promoter! vector - Targeting signal - Purification tag ■ Strain o Optimization - Temperature - Promoter / vector - Induction - Cullure media Optimization - Cullure MTiaiiiong - Signal sequence - Gene dosage - Strain "ft. Optimization - Temperature -pH ■ Osrmolarily - Aeration - Shear forces 4 Protein purification Tflf fjp$ in Parasitology https://www.cell.com/trends/parasitology/fulltext/S1471 -4922(10)00025-5 le of interest, targeting and selection of noter, into a that can be Dale needed elude 3specting the jsidues at the antigenicity or organism or i suite of 82 LOSCHMIDT LABORATORIES CRISPR/Cas9-directed evolution (CDE) in plants 3 Design of targeted sgRNA library b Construction of sgRNA library C Cloning in binary vector d Plasmid pooling Variant WT ATAAATCTGGAGTTA WT protein Protein variant m I Genotyping 15-GGCANNNNNNNNNNNNNNNNNNNN-3'_S S'-GGCANNNNNNNNNNNMNNMNNNNN^' | 5'-GGCANNNNNNNNNNNNNNNNNNNN-3' I I I I I I I I I I I I I I I I I I I I 5'NNNNNNNNNNNNNNNNNNNNCAAA 3 v- J WT Variant h Phenotyping under selective pressure 9 Regeneration under selective pressure leAgrobacterium transformation f Callus transformation by agrobacterium 83 , LOSCHMIDT ; LABORATORIES A GUIDE TO THE TWENTY COMMON AMINO ACIDS AMINO ACIDS ARE THE BUILDING BLOCKS OF PROTEINS IN LIVING ORGANISMS. THERE ARE OVER 500 AMINO ACIDS FOUND IN NATURE - HOWEVER, THE HUMAN GENETIC CODE ONLY DIRECTLY ENCODES 20. 'ESSENTIAL'AMINO ACIDS MUST BE OBTAINED FROM THE DIET, WHILST NON-ESSENTIAL AMINO ACIDS CAN BE SYNTHESISED IN THE BODY. Chart Hey: ALIPHATIC AROMATIC ACIDIC BASIC HYDROXYLIC SULFUR-CONTAINING AMI DIC O NON-ESSENTIAL I I ESSENTIAL name O three tetter cade UNA cottons alanne0 Ala tit I, lit!, litA, ütli glycine G Gly Mil. üüt.ütiA, ütiS isoleugineO lie Al I.Alt, A1A NH \ \ OH I leucine O Leu t ii. tit, tlA. t 10 i ia i It. proline O Pro CCl. ttt, Ltfi, ttü I OH I NH, \ / _ *<* valine O Va! ÜI l.tjlt. O'VUt phenylalanine Phe TTT.TTf tryptophan 0 Trp Ter, tyrosine 7yr aspart1cacidq glutamic acid O tat, tac c;atp gat r~*A,r.AT, arginine O Arg ca, rr.c, rr.A, rer.. .'«"„',. A;",r. / 1 € ii ^ HN-\ \ ° \ OH I histidine O \ o \ OH lysine O AAA, AAC serine 0 TfT, T This chart only shows those amino acids for which the human genetic code directly codes for. Selenocysteine is often referred to as the 21st amino acid, but is encoded in a special manner. In some cases, distinguishing between asparagine/aspartic acid and glutamine/glutamic acid is difficult, In these cases, the codes asx (B) and glx (Z) are respectively used. ©COMPOUNDINTEREST2014-WWW.COMPOUNDCHEM.COM | Twitter: @compoundchem | Facebook: www.facebook.com/compoundchem Shared under a Creative Commons Attribution-Noncommercial NoDcrivativcs licence. BY HC HQ