Transcription

GeneArt Gene to ProteinYour gene, your way.

ContentsGene to protein1Mammalian expression systemsChoosing an expression system3Selecting a mammalian expressionsystem13Expi expression systems15Building your geneGeneArt Gene Synthesis5Optimizing expression6GeneArt Strings DNA Fragments8Choice of your vector9Choice of cloning kits10Subcloning options12Protein production services18Creating improved proteinsGeneArt Directed Evolution20GeneArt Strings DNA Libraries21Find out more at thermofisher.com/geneartproducts

Gene to proteinFrom creating your gene to expressing your protein, we offer a wide range of tools and expression systems thatare easy to use, enable rapid results and offer high protein yields for every downstream application (Figure 1).This brochure outlines our recommended products and services for your research needs, which can help saveyou weeks of hands-on time compared to using traditional methods. We also offer a complete service for theentire gene-to-protein workflow (page 18).Creating constructRestriction enzymes,GeneArt Seamless Cloningor Type IIs Assembly kits,TOPO cloning, orGateway kitsPlasmid purificationDeliveryKingFisher MagneticParticle ProcessorsLipofectamine 3000 reagentGeneArt StringsDNA FragmentsPureLinkHiPure plasmid kitsLipofectamine LTX ReagentGeneArtGene Synthesis servicesand subcloningGeneArt plasmidpurification servicesNeon Transfection SystemGene synthesisPlasmid purificationExpressionExpi293 and ExpiCHOsystemChampion pET systemPichiaPink systemBaculovirus systemGeneArt algae systemOther systemDeliveryExpressionGeneArt Genes-to-Proteins serviceFigure 1. General workflow for protein expression.1

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Choosing an expression systemRecombinant proteinexpression technologyis essential for a widevariety of applications.From the study of proteinfunction to the large-scaleproduction of proteinsfor drug discovery anddevelopment, using theright expression systemfor your application isimportant for success.Protein solubility,functionality, purificationspeed, and yield areoften crucial factors toconsider when choosingan expression system.With our wide variety ofexpression systems, you’resure to find one that meetsyour needs (Table 1). Thefollowing pages focus onbuilding and cloning optionsfor your gene and providedetails on mammaliansystems to enhance yourexpression.Table 1. Protein expression systems and their applications. Each system has its own strengths and challenges,which are important to consider when choosing an expression system.HostorganismMost commonapplicationsAdvantagesChallengesProkaryotic Structural analysis Scalable Protein solubility Antibody generation Low cost Functional assays Simple culture conditions Minimal posttranslationalmodifications Protein interactions Compatible with Gateway cloning Structural analysis Eukaryotic protein processing Antibody generation Scalable up to fermentation(grams/liter)Yeast Functional assays Protein interactionsInsect Functional assays Structural analysis Antibody generation Simple media requirements Posttranslational modificationssimilar to mammalian systemsFor n May be difficult toexpress functionalmammalian proteins Fermentation requiredfor very high yieldsthermofisher.com/yeastexpression Growth conditions mayrequire optimization More demandingculture conditionsthermofisher.com/insectexpression Multimilligram/literyields only possible insuspension culturesthermofisher.com/mammalianexpression Usually greater yields than frommammalian systems Compatible with Gateway cloningMammalian Functional assays Protein interactions Antibody generation Highest level of correctposttranslational modifications Highest probability of obtainingfully functional human proteins Compatible with Gateway cloningAlgal Basic algal research Plant sciences Protein production More demandingculture conditions Fast-growing, photosyntheticmodel organism Difficulty preserving andreviving cells Superb experimental controlfor biofuels, nutraceuticals, andspecialty chemical production Silencing of expression ofgene of interest Optimized system for robustselection and expressionthermofisher.com/algaeexpression Long growth cycle ofphotosynthetic land plantsFor a complete list of our expression systems, please go to thermofisher.com/proteinexpress3

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Building your geneGeneArt Gene SynthesisGene synthesis has become the most cost-effective and time-savingmethod for obtaining nearly any desired DNA construct. Simply providethe sequence you want, and the gene will be synthesized and cloned foryou (Figure 2). When combined with gene optimization, gene synthesisproduces clones that outperform constructs made using conventionalmolecular biology techniques. Invitrogen GeneArt Gene Synthesis toolsgo beyond traditional cloning and enable you to:Beyond gene synthesis, we also offer Invitrogen GeneArt Strings DNA Fragments, which are delivered as linear double-stranded DNAfragments. If you prefer to synthesize your own genes, you can usethe Invitrogen GeneArt Gene Synthesis Kit, which provides all of thehigh-quality reagents necessary for successful production of syntheticfragments. Table 2 gives a summary of our gene synthesis options. Improve protein expression with Invitrogen GeneOptimizer technologyAGwww Gain access to hard-to-clone constructs with long, complex DNAGAAGG5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘A C G C A Overcome gene or vector design limitationsDay 1Ordering until3:00 p.m. (CET) Create unlimited numbers of mutants for screening experimentsOligo synthesisovernightDay 2GeneAssemblerprocessDay 3CloningDay 4Sequencing andquality controlDay 5Ready forshipmentFigure 2. Invitrogen GeneArt SuperSPEED production schedule. Genes can besynthesized, cloned, and shipped in as few as 5 business days. Engineer proteins to improve enzyme activity and increase bindingaffinities of antibodiesTable 2. Gene synthesis options.Do-it-yourself gene synthesisCustom DNA fragmentsCustom gene synthesisProduct or serviceGeneArt Gene Synthesis KitGeneArt Strings DNA FragmentsGeneArt Gene Synthesis andSubcloning ServiceAdvantage Full cost control Fast and affordable 100% sequence verified You control every step Design flexibility Convenient ordering Gene optimization Design flexibility No physical template required Gene optimization Reliable technology available toassemble your complete gene (e.g.,GeneArt Seamless PLUS Cloning andAssembly Kit) No physical template required No synthesis and cloning hands-ontime needed Optional service speed upgrades(e.g., SuperSPEED [see Figure 2])Lab workHighMediumLowStandard processingtimeNA5 business days for fragments up to1,000 bp and 8 business days forfragments up to 3,000 bpFrom 9 business days for genes up to1,200 bpOrder genes on our easy-to-use portal at thermofisher.com/genesynthesis5

Optimizing expressionOptimization of your gene sequence can be beneficial from severalperspectives. Production of recombinant proteins for biomedical researchand product development can be hampered by low expression yields.These expression issues can limit the ability of researchers to conductstructural and functional analyses, delaying, and in some cases haltingthe discovery process. Gene optimization is the solution to traditionalprotein expression limitations. The common challenges associated withprotein expression—yield, solubility, and functionality—can now beaddressed in a rational and systematic way.Secondly, if you consider total costs for your expression experiments, anoptimized gene with higher expression rates can save you money. Withhigher expression rates, you might consider to scale down experimentalsetup and save on costs for materials (e.g., media, vessels).Remove sequencerepeatsPABPThirdly, gene optimization can help you to also obtain genes, which, due toinstability elements in the wild type sequence, might not be efficiently cloned byother cloning methods.How does it work?The Invitrogen GeneOptimizer algorithm determines the optimal gene sequencefor your expression system as part of a real multiparametric approach, which isnot just simply a codon optimization process (Figure 3). By evaluating the relevantexpression parameters in parallel, GeneOptimizer technology generates a plethoraof variants of your target sequence in an evolutionary approach and selects thebest match for your specific requirements. Sequence optimization using theGeneOptimizer software process is included as an optional step with all GeneArtGene Synthesis services and with GeneArt Strings DNA Fragments.Adjust codon usageOptimize GC contentPABPPABPEliminate killer motifsAAAAAAAAAAAARemove splice sitesAvoid RNA secondarystructureOptimized geneFigure 3. The GeneOptimizer algorithm determines the optimal gene sequence for your experiments. Thealgorithm takes many factors into account—for example, it removes DNA sequence repeats, optimizes codon usage andGC content, and minimizes the formation of RNA secondary structures that may reduce protein yield. Protein sequence isnot affected by the optimization process.6

Relative expression60CREB1x 2.8Relative exressionIn a first-of-its-kind study [1], five important protein classes were selectedCREB135optimization—protein kinases, transcriptionforfactors, ribosomal proteins,cytokines, and membrane proteins. Then, 50 human genes were chosen fromWildselectedtype genesOptimizedthe NCBI database to represent the five protein classes. Thewereindividually optimized using the GeneOptimizer algorithm [2]. For comparison, theSMARCD1corresponding wild type genes were subcloned using native sequences availablex 1.8from the NCBI database. Each gene was then expressed in triplicate inkDaHEK 293T cells. Following optimization, the 50 genes all showed reliable60expressionand 86% exhibited elevated expression (example in Figure 4).SMARCD1Furtheranalysisshowed no detrimental effect on protein solubility, and unaltered40functionality was demonstrated for JNK1, JNK3, and CDC2 (data not shown).Wild typeOptimizedUsing the GeneOptimizer algorithm, in this study:Relative exression 86% of optimized genes showed significantly increasedprotein expressionkDaJNK3x 15.0 Protein yields increased up to 15-fold with optimized genes60 100% of optimized genes were expressed,versus 88% ofJNK3wild type genes40PP3PP2OptimizedPP1PP3PP2PP1kDaMockWild type6030AQP5Wild typeOptimizedOptimizedRelative exressionFigure 4. Comparative expression analysis of wild type vs. the optimized gene for aquaporinIL-25, an integral membrane protein. (A) Cell lysate was analyzed by western blot using an anti-Hisx only optantibody. A 60 kDa protein used to standardize protein amount is visible. (B) Expression levels werekDaderived20 for the wild type and optimized construct (mean of 3 independent transfections) and therelative expression from each construct is indicated. Figure adapted from Fath et al., 2011 eWildtypeGeneArtGeneArtGenscript 1 CompetitorDNA2.0 2 CompetitorBlue Heron 3 CompetitorIDTGenewiz 5Competitor4 CompetitorFigure 5. Expression levels from wild type genes and the same genes optimized by GeneArttechnology and five competitors. Relative protein expression values are normalized to therespective GeneArt sequence.Wild type151.00.2Relative expressionBATo continue our GeneOptimizer gene optimization success story and demonstrateits true value, we performed a large-scale competitor study. Genes for 3 differenthuman kinases were optimized and synthesized in-house or by 5 differentcompetitors. Triplicate expression studies in HEK 293 cells not only show thatthe GeneArt optimization raises expression over the wild type genes, but alsoperforms better than any competitor’s optimization algorithm in every case(Figure 5).Rrelative protein expressionPP3PP2PP1OptimizedPP3PP2PP1MockkDaWild typeReferences1. Fath S, Bauer AP, Liss M et al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess andenhance autologous mammalian gene expression. PLoS One 6:e17596.2. Raab D, Graf M, Notka F et al. (2010) The GeneOptimizer algorithm: using a sliding window approach to cope with the vastsequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4:215–225.Find out more about gene optimizationat thermofisher.com/geneoptimizationOptimizedWild type7

GeneArt Strings DNA FragmentsGeneArt Strings DNA Fragments are custom-made linear double-stranded DNA fragmentsup to 3 kb. A quantity of at least 200 ng of GeneArt Strings DNA Fragments is producedwithin 5 business days, ready for cloning in your lab. GeneArt Strings DNA Fragments canbe ordered online and offer a fast, convenient, and cost-effective alternative to traditionalPCR-based cloning.GeneArt Strings DNA Fragments are produced with the same technology used for GeneArtGene Synthesis. DNA fragments are assembled from high-quality synthetic oligonucleotidesand bulk sequenced to verify that your desired gene is highly represented in the fragmentpool.Experimental data (Figure 6) show a high probability of finding a correct clone if yousequence: 2–4 full-length clones for Strings DNA Fragments 1 kb 3–5 full-length clones for Strings DNA Fragments 1–2 kb 4–8 full-length clones for Strings DNA Fragments 2–3 kbWe offer several reliable and efficient Invitrogen cloning systems, such as restrictionenzymes, TOPO cloning, Gateway cloning, GeneArt Seamless Cloning, and GeneArt Type IIs Assembly Kits (see page 10). We recommend that you first choose a suitablecloning system and then design your Strings DNA Fragments according to the respectiverequirements. Cloning efficiency9897%Success rate (%)See how GeneArt Strings DNAFragments outperformed IDT gBlocks Gene Fragments.WHITE PAPERGeneArt Strings DNA Fragments vs. gBlocks Gene FragmentsIntroductionGene synthesis has become an indispensable tooland reliable source of genetic material for the researchcommunity. The process of de novo gene synthesisinvolves assembly and PCR amplification of chemicallysynthesized oligonucleotides. Since this chemistry isnot entirely perfect, a small amount of sequence erroroccurs during oligonucleotide synthesis that remainsthrough the downstream assembly process. Cloningand sequencing, therefore, is a reasonable way toscreen for error-free molecules and discard thosewith mutations.Download the white paper 00 bpFigure 6. Cloning Strings DNA Fragments using restriction enzyme cloning. The success rateof conventional restriction enzyme cloning was evaluated with more than 1,000 fragments directlytaken from our standard gene synthesis manufacturing process. Fragments of different size ranges,100–500 bp and 501–1,000 bp, were subjected to restriction enzyme cloning. Bacterial colonies wereanalyzed by colony PCR to determine cloning efficiency and then sequenced. Sequencing of up to 4full-length fragments between 100 bp and 500 bp and up to 6 full-length clones between 501 bp and1,000 bp resulted in success rates of 90% and 85%, respectively, to find correct clones.501–1,000 bpFragment sizeFind out more at thermofisher.com/stringsFigure 1. Possible applications for linear synthetic DNA fragments.GeneArt Strings DNA Fragments arealso available as GeneArt StringsDNA Libraries (see page 21 for moredetails).Correct sequence90%After screening, synthetic genes are typically deliveredin a cloned and sequenced plasmid to ensure 100%sequence accuracy. However, many scientists preferto directly obtain linear synthetic DNA fragments toperform cloning and sequencing on their own or for avariety of other applications, including direct assemblyand cloning for protein expression, CRISPR-basedgenome editing, in vitro transcription and translation,or real-time PCR (Figure 1).Did you know?9490GeneArt Strings DNA FragmentsEvaluation of linear synthetic DNAfragments from separate suppliers96%968GeneArt Strings DNA white paper

Choice of your vectorGeneArt Elements Vector ConstructionThe choice of the appropriate expression vector is essential for thesuccess of your protein expression experiments. We offer a broad varietyof expression vectors for all common expression systems, which can befound using the Vector Selection Tool at thermofisher.com/vectorsIf your required vector is not commercially available, you can design andcreate your individual vector using our Invitrogen GeneArt Elements Vector Construction services. We offer a growing collection of biologicallywell-characterized components, such as promoters, terminators,enhancers, operators, and open reading frames, with exactly definedsequences and functionalities (Table 3). These predefined parts can beindividually combined with custom components to build your favoritevector. Our online portal features in vitro assembly of predefined andcustom components without any limitations from the DNA sequences atthe junctions. The designed vector can be directly ordered via our onlineorder portal.GeneArt online portalTable 3. Examples of elements that can be used to construct customized vectors.Part numberPublic namePart family/class Part categoryGA-pro-00016.1CMV ctionrestriction siteGA-rep-00001.1emerald GFPCDSreporterGA-rep-00002.1YFP riction siteGA-clv-00002.1TEV recognitionsiteCDSproteasecleavage siteGA-tag-00002.16x His tagCDStag (purification,detection)GA-cod-00063.1Stop codon TAACDScodonGA-pla-00001.1BGH polyA siteregulationpolyA siteGA-ori-00002.1f1 originregulationreplication originGA-pro-00020.1SV40 promoterregulationpromoterGA-cod-00001.1Start codonCDScodonGA-tag-00006.1V5 tagCDStag (purification,detection)GA-tag-00001.1c-myc tagCDStag stancecassette formammalian cellsCDSresistancecassetteNeomycinresistance ORF;npt2CDSresistance geneCDStag (purification,detection)Use our online portal for easy andconvenient ordering.Get started GA-tag-00007.1HA tagAdditional parts can be found at thermofisher.com/geneartelementsvc9

Choice of cloning kitsThere is broad range of cloning technologies available to clone your GeneArt Strings DNA Fragment or gene synthesis product into your favoritevector yourself. We have developed a variety of easy-to-use cloning kits to meet your needs (Table 4).Table 4. Suitable cloning systems for GeneArt Strings DNA Fragments and gene synthesis nTechnical resourcesCloning by restriction enzyme digestion and ligation is a simple and easy way of moving a fragment ofdouble-stranded DNA from one plasmid to another.thermofisher.com/restrictionGeneArt Strings DNA Fragments or gene synthesis products must have the restriction enzyme sitesincorporated at the ends of the sequence, plus stuffer nucleotides. Continue cloning via standardrestriction enzyme and DNA ligase technology.GeneArtType IIsAssemblykitsAssembly with Type IIs restriction enzymes and DNA ligase, also known as Golden Gate cloning, canbe used to assemble up to eight DNA fragments. It is highly efficient and is not based on homologousrecombination. Consequently, it is less prone to unwanted rearrangements due to repetitive orhomologous sequences and minimizes the need for sequence confirmation of your final construct.thermofisher.com/typeiisGeneArt Strings DNA Fragments or gene synthesis products must have appropriate restriction enzymesites incorporated at the ends (either AarI, BsaI, or BbsI, depending on the kit), plus stuffer nucleotides.For more detailed information, refer to the GeneArt Type IIs Assembly Kit manual, “Guidelines forGenerating DNA Inserts” (Cat. Nos. A15916, A15917, and A15918).GeneArtSeamlessCloning andAssemblykitsGeneArt Seamless Cloning and Assembl