Gene To Protein - Thermo Fisher Scientific

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Gene to proteinEngineer innovation

ContentsGene to protein1Choosing an expression system3Selecting a mammalian expression system 10Building your gene5Expi293 Expression System12GeneArt Gene Synthesis5GeneArt Algae Engineering Kits15Optimizing expression6GeneArt Strings DNA Fragments8Gateway technology for multipleexpression systems17Gene assembly9Protein production services18Creating improved proteins20Mammalian expression systemsGeneArt Directed Evolution1020Find out more at lifetechnologies.com/proteinexpress

Gene to proteinFrom creating your gene to expressing your protein, Life Technologies offers a wide range of toolsand expression systems that are easy to use, enable rapid results, and offer high protein yields forevery downstream application (Figure 1). This brochure outlines the best products and services foryour research needs, which can help save you weeks of hands-on time compared to using traditionalmethods. We even offer a complete service for the entire gene-to-protein workflow (page 18).CloningPlasmid purificationDeliveryExpressionExpi293 systemGateway , TOPO , orType IIs cloningBenchPro 2100Lipofectamine 2000Lipofectamine LTXGeneArt gene synthesisPureLink HiPure kitsNeon Transfection SystemChampion pET systemPichiaPink systemBaculovirus systemAlgae or other systemGene synthesisPlasmid purificationDeliveryExpressionGeneArt Gene-to-Proteins serviceFigure 1. General workflow for protein expression.Life Technologies Gene to protein1

Choosing an expression systemRecombinant proteinexpression technologyis essential for a widevariety of applications.From the study ofprotein functionto the large-scaleproduction of proteinsfor drug discovery anddevelopment, usingthe right expressionsystem for yourapplication is importantfor success. Proteinsolubility, functionality,purification speed, andyield are often crucialfactors to consider whenchoosing an expressionsystem. With the widevariety of expressionsystems available fromLife Technologies, you’resure to find one thatmeets your needs(Table 1). The followingpages detail mammalianand algal tools toenhance your expression.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 May be difficult toexpress functionalmammalian proteins Structural analysis Eukaryotic protein processing Antibody generation Scalable up to fermentation(grams/liter) Fermentation requiredfor very high nlifetechnologies.com/yeastexpression Protein interactions Simple media requirements Growth conditions mayrequire optimization Functional assays Posttranslational modificationssimilar to mammalian systems More demandingculture conditionslifetechnologies.com/insectexpression Multimilligram/literyields only possible insuspension cultureslifetechnologies.com/mammalianexpression Functional assaysInsectFor more information,go to: Structural analysis Antibody generation Usually greater yields than frommammalian systems Compatible with Gateway cloningMammalian Functional assays Protein interactions Antibody generationAlgal Basic algal research Plant sciences Protein production Highest level of correctposttranslational modifications Highest probability of obtainingfully functional human proteins Compatible with Gateway cloning 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 expressionlifetechnologies.com/algaeexpression Long growth cycle ofphotosynthetic land plantsFor a complete list of expression systems from Life Technologies,please go to lifetechnologies.com/proteinexpressLife Technologies Choosing an expression system3

Building your geneGeneArt Gene SynthesisGene synthesis has become the most cost-effective and timesaving method for obtaining nearly any desired DNA construct.Simply provide the sequence you want, and the gene will besynthesized and cloned for you (Figure 2). When combined withgene optimization, gene synthesis produces clones that outperformconstructs made using conventional molecular biology techniquesin expression performance. GeneArt Gene Synthesis tools gobeyond traditional cloning and enable you to: Improve protein expression with GeneOptimizer technologyGain access to hard-to-clone constructs with long, complex DNAOvercome gene or vector design limitationsCreate unlimited numbers of mutants for screening experimentsEngineer proteins to improve enzyme activity and increasebinding affinities of antibodiesBeyond gene synthesis, Life Technologies also offers GeneArt Strings DNA Fragments, which are delivered as linear, doublestranded DNA fragments. If you prefer to synthesize your owngenes, you can use the GeneArt Gene Synthesis Kit, whichprovides all of the high-quality reagents necessary for successfulproduction of synthetic fragments. Table 2 provides a summary ofgene synthesis options available from Life Technologies.AGwwwGAAGG5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘A C G C ADay 1Ordering until3:00 pm (CET)OligosynthesisovernightDay 2GeneAssembler processDay 3CloningDay 4Sequencing &quality controlDay 5Ready forshipmentFigure 2. GeneArt SuperSPEED production schedule. Genes can be synthesized,cloned, and shipped in as few as 5 business days.Table 2. Gene synthesis options from Life Technologies.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 to assembleyour complete gene (e.g., GeneArt Seamless PLUS Cloning and Assembly Kit) No synthesis and cloning hands-ontime needed Optional speed upgrades(e.g., SuperSPEED (see Figure 2))Lab workHighMediumLowStandard processing timeNA5 business days for fragments up to 1,000 bp9 business days for genes up to 1,200 bpOrder genes on our easy-to-use portal at lifetechnologies.com/genesynthesisLife Technologies Building your gene5

Optimizing expressionProduction of recombinant proteinsfor biomedical research and productdevelopment can be hampered by lowexpression yields. These expressionissues can limit the ability of researchersto conduct structural and functionalanalyses, delaying and in some caseshalting the discovery process. Geneoptimization is the solution to traditionalprotein expression limitations. Thecommon challenges associated withprotein expression—yield, solubility, andfunctionality—can now be addressed in arational and systematic way.The GeneOptimizer algorithm determinesthe optimal gene sequence for yourexpression system as part of a realmultiparametric approach (Figure 3).By evaluating the relevant expressionparameters in parallel, GeneOptimizer technology generates a plethora ofvariants of your target sequence in anevolutionary approach and selects thebest match for your specific requirements.Sequence optimization using theGeneOptimizer software process isincluded as an optional step with allGeneArt Gene Synthesis Services andwith GeneArt Strings DNA Fragments.6Life Technologies Gene to proteinRemove sequencerepeatsPABPAdjust codon usageOptimize GC contentPABPPABPEliminate killer motifsAAAAAAAAAAAARemove splice sitesAvoid RNA secondarystructureOptimized geneFigure 3. The GeneOptimizer algorithm determines the optimal gene sequence for your experiments.The algorithm removes DNA sequence repeats, optimizes codon usage and GC content, and minimizes theformation of RNA secondary structures that may reduce protein yield. Protein sequence is not affected by theoptimization process.

1Relative exressionBWild TypeCREB1x 2.8Relative exressionASMARCD1x 1.860JNK340Relative exressionwild typeJNK3x 15.030AQP5Relative exressionwild type60IL-2Relative exression15optimizedAQP5x 9.0wild type20optimizedoptimizedIn a first-of-its-kind study [1], five important protein classes wereselected for optimization—protein kinases, transcription factors,ribosomal proteins, cytokines, and membrane proteins. Then, 50human genes were chosen from the NCBI database to representthe five protein classes. The selected genes were individuallyoptimized using the GeneOptimizer algorithm [2]. For comparison,the corresponding wild type genes were subcloned using nativesequences available from the NCBI database. Each gene was thenexpressed in triplicate in HEK 293T cells. Following optimization,the 50 genes all showed reliable expression and 86% exhibitedelevated expression. Further analysis showed no detrimental effecton protein solubility, and unaltered functionality was demonstratedfor JNK1, JNK3, and CDC2 (data not shown). Using the GeneOptimizer algorithm, in this study: 86% of optimized genes showed significantly increasedprotein expression Protein yields increased up to 15-fold with optimized genes 100% of optimized genes were expressed, versus 88% ofwild type genesIL-2x only optwild typeoptimizedFigure 4. Comparative expression analysis of wild type versus optimized genesrepresenting different protein classes. (A) Cell culture supernatants (for secretedproteins) or cell lysates (all other proteins) were analyzed by western blots using ananti-His antibody. One example of each protein class is shown. A 60 kDa protein used tostandardize protein amount is visible, including in the empty vector negative controls. Leftof each image: molecular mass values in kDa. Right of each image: identifiers for specificprotein bands. (B) Relative expression levels were derived for wild type or optimizedconstructs (mean of three independent transfections). The fold increase in expression forthe optimized construct is indicated for each protein. There was no detectable expressionfor IL-2 using the wild type construct. Figure adapted from Fath et al., 2011 [1].References1. Fath S, Bauer AP, Liss Met al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologousmammalian gene expression. PLoS One 6:e17596.2. Raab D, Graf M, Notka Fet al. (2010) The GeneOptimizer algorithm: using a sliding window approach to cope with the vast sequence space inmultiparameter DNA sequence optimization. Syst Synth Biol 4:215–225.Find out more about gene optimization atlifetechnologies.com/genesynthesisLife Technologies Building your gene7

GeneArt Strings DNA FragmentsGeneArt Strings DNA Fragments are custom-made linear double-stranded DNAfragments. A quantity of at least 200 ng of GeneArt Strings DNA Fragments isshipped within 5 business days, ready for cloning in your lab. GeneArt Strings DNA Fragments can be ordered online and offer a fast, convenient, and affordablealternative to traditional PCR-based cloning.GeneArt Strings DNA Fragments are produced with the same technology used forGeneArt Gene Synthesis. DNA fragments are assembled from high-quality syntheticoligonucleotides and bulk sequenced to verify that your desired gene is highlyrepresented in the fragment pool.Experimental data show a high probability of finding a correct clone if you sequenceat least 4 colonies containing full-length inserts (Figure 5). Life Technologies offersseveral reliable and efficient cloning systems, such as TOPO cloning, Gateway cloning, GeneArt Seamless Cloning, and GeneArt Type IIs Assembly Kits. Werecommend that you first choose a suitable cloning system and then design yourStrings DNA Fragments according to the respective requirements.Cloning efficiency98Success rate (%)96Looking for the rightkit to isolate plasmidDNA at the purity andscale you need?Go to lifetechnologies.com/plasmidprepCorrect sequence97%96%94929090%888685%84828078100–500 bpFigure 5. Cloning Strings DNA Fragments using restriction enzyme cloning. The success rate ofconventional restriction enzyme cloning was evaluated with more than 1,000 fragments directly takenfrom 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 4 fulllength fragments between 100 bp and 500 bp and up to 6 full-length clones between 501 bp and 1,000 bpresulted in success rates of 90% and 85%, respectively, to find correct clones.501–1,000 bpFragment size8Plasmid isolationLife Technologies Gene to proteinFind out more at lifetechnologies.com/strings

Gene assemblyGeneArt Type IIs Assembly KitsGeneArt Type IIs Assembly Kits provide seamless cloning andassembly of up to 8 DNA fragments by simultaneous cleavageand ligation in a single reaction (Figure 6). The kits use atechnology similar to Golden Gate cloning with Type IIs restrictionenzymes, and can be used to assemble multiple fragments ina predetermined order into any compatible vector. Since TypeIIs assembly is not based upon homologous recombination,there is minimal risk of rearrangements, and minimal sequenceconfirmation of your final construct is required. Life Technologiesoffers three kits, each with one of three Type IIs restrictionenzymes: AarI, BsaI, and BbsI, all of which come with an all-i

GeneArt Strings DNA Fragments 8 Gene assembly 9 Mammalian expression systems 10 Selecting a mammalian expression system 10 . the five protein classes. The selected genes were individually optimized using the GeneOptimizer algorithm [2]. For comparison, the corresponding wild type genes were subcloned using native sequences available from the NCBI database. Each gene was then .

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