expression solutions VectorGPS(R) Expression vectors Electra cloning system GeneGPS optimization Gene synthesis & custom cloning expression Expression of active protein at high levels requires selecting the best coding sequence for a gene and the best vector from which to express it. ATUM’s empirically-derived codon optimization ensures that your gene is designed for efficient translation in your chosen host. Our expression vectors combine control elements that work well together for mammalian, bacterial or yeast systems. For especially challenging proteins or new host systems, we can develop customized solutions. We can even test the expression or produce the protein for you. ATUM has developed a variety of expression vectors, using Design-of- Experiment techniques to test interactions between control elements, secretion signals and functional tags. Vector elements also interact with the gene being expressed, so there is no one vector that will work for all proteins. We have selected the vectors in our catalog to maximize the chances that at least one will have the expression properties you need. contents Mammalian Expression: Vectors and Services 2-11 Transient, stable, Leap-In transposase and lentivirus vectors for high protein expression. Bacterial Expression Vectors 12-13 High protein expression, with a choice of promoters, markers, tags, secretion signals and more. Yeast Expression Vectors 14-15 High protein expression, with a choice of promoters, markers, tags, secretion signals and more. Electra Vector System(R) 16 One tube, 5 minutes, any ORF. Scarless cloning in bacterial, mammalian and yeast systems. GeneGPS(R) Optimization 17 One tube, 5 minutes, any ORF. Scarless cloning in bacterial, mammalian and yeast systems. Expression Services 18 Gene synthesis, Custom Cloning, LSPs, and Expression Test. VectorGPS(R) 19 Discover the vector that works best for your research. mammalian expression: vectors & services ATUM offers vectors for transient and stable 70 mammalian protein expression. Vectors shown here are available from the ATUM catalog as easy 60 to clone Electra kits. Any synthetic gene made by pD2610-v1 ATUM can also be cloned into these vectors. 50 pD2610-v12 pD2610-v26 40 pD2600 Transient Expression Vectors pD2610-v10 pD2610-v14 We offer around 30 different transient vector 30 configurations, which we identified by testing over pD2610-v13 pD2610-v9 pD2610-v3 250 different combinations of promoters, introns, 20 pD2610-v4 pD2610-v23 pcDNA3.4 pTT5 pD2610-v27 polyadenylation sequences and viral amplifiers in pD2610-v2 pD2610-v5 pD2610-v28 HEK and CHO cell lines. pD2610-v6 10 pD2610-v11 pD2610-v29 pD609 pD2610-v17 HEK293 Expression (Fluorescence X1000) pD2610-v19 pD2610-v16 Intron ORF pD2610-v8 pD2610-v20pD2610-v18 0 pD2610-v7 pD2610-v15 0 10 20 30 40 50 CHO-K1 Expression (Fluorescence X1000) polyA Globin BGH... Promoter CMV 120 EF1a GAPDH... pD2610-v5 Enhancer pD2600 100 CMV EF1a pD2610-v2 SV40 Synthetic... pD2610-v1 80 pcDNA3.4 pD2610-v14 pD2610-v13 SV40 ori EBV oriP pD2610-v12 pD2610-v23 SV40 large T-Ag pTT5 pD2610-v2p6D2610-v4 pD2610-v10 Bacterial resistance marker EBNA... 60 pD2610-v9 Ori pD2610-v3 pD2610-v29 The reason we have so many vectors is that even 40 pD2610-v27 similar cell lines seem to have different control pD2610-v11 Expression (Fluorescence X1000) pD2610-v30 element preferences, as is shown in the graphs TM 20 pD2610-v17 below. Added to this, differences in desired pD2610-v15 pD2610-v6 duration of culture and the localization of the Expi293 0 pD2610-v28 protein being produced (intracellular, membrane- 0 10 20 30 40 50 60 70 bound or secreted) make it impossible for a single Expi-CHOTM Expression (Fluorescence X1000) vector to meet all needs. DasherGFP expression compared in transiently transfected CHO and HEK293 cells. Cells were harvested from independent triplicate transfections and DasherGFP fluorescence was measured 72 hours post-transfection. 2 www.atum.bio for your protein, please call us! Or let us make the protein for you. for the protein us make let callus!Or please protein, your for need helpinchoosingavector you soif andCHO, inHEK week every proteins of hundreds express We anIRES. through coupling expression chainsby and light heavy antibody’s of co-expression enable construct) (single CHO cells. configurations vector dualexpression ATUM and HEK293 transfected transiently in production Antibody Vectors Expression pD2600 Transient HEK Expression - Antibody conc. in µg/ml 30 40 50 20 35 45 25 -5 10 15 50 0 0 0 0 0 0 0 0 0 0 0 pD2610-v1 pD2610-v2 pD2610-v3 pD2610-v4 pD2610-v5 pD2610-v6 pD2610-v7 pD2610-v8 pD2610-v9 chain quantitation by ELISA, 6 days post-transfection. post-transfection. 6days ELISA, by chain quantitation heavy by measured was expression Antibody cells. CHO and inHEK293 triplicate carriedout in were transfections Fugene; using confluency at70-80% transfected Cells were pD2610-v10 pD2610-v11 MM Ve Ri tu pD2610-v12 ct 12 xa ib 1V ix n pD2610-v13d pD2610-v13 He He 23 pD2610-v14 LN rc rc ep ep pD2610-v15 ti ti MAMMALIAN EXPRESSION n n pD2610-v16 (A TU pD2610-v17 M co pD2610-v18 do n 3 op pD2610-v19 ti mi pD2610-v20 ze d) transient expression services Protein Expression Whether you need milligrams of thousands of Our versatile platform produces proteins including proteins as part of a drug candidate screening antibodies, Fabs and Fc fusions at 1ml - 20l scales. process, or grams of a few proteins for in depth Delivery times are accelerated because sequence characterization of your best leads, we can help. and vector optimization, synthesis & cloning, expression and purification are all under one roof. An automated, bar-coded, time-stamped process can screen thousands of expressed proteins in parallel. For large screening projects, it can be worth spending time optimizing the components. This can include: ⬤ Optimizing codon bias and eliminating potentially problematic cryptic splice sites from DNA sequences. ⬤ Selecting efficient signal sequences for secreted proteins. ⬤ Identifying the best vector. ⬤ Balancing chain ratios for antibodies or other proteins with multiple polypeptides. 4 www.atum.bio MAMMALIAN EXPRESSION The graph shows expression 250 of twelve representative murine antibody sequences 200 for a screening project. The genes were cloned into 5 different vectors, transfected 150 into HEK cells and harvested 5 days later. Based on this Titer in mg/l 100 data we selected vector #2 for further process 50 development. 0 1 2 3 4 5 Vectors After optimizing secretion 250 signals and constant region coding region as well as the l vector sequence, we were 200 able to exceed the target productivity of 75 mg/l 150 in >95% of the expressed antibodies. 100 Antibody Yield in mg/ 50 0 1 1 1 7 5 9 3 3 9 61 13 97 37 73 25 49 12 85 18 15 14 16 19 13 10 Constructs Proteins are delivered with a rigorous QC package. ⬤ Protein concentration (A280) ⬤ Molecular weight (reducing and non-reducing gel electrophoresis) ⬤ Aggregation (size exclusion chromatography) And optionally: ⬤ Endotoxin (Charles River test) ⬤ Glycan analysis (mass spectroscopy) ⬤ Binding affinity (Surface plasmon resonance with the Octet) 5 pD2500 & pD3600 stable vectors ATUM offers two stable vector series: the pD2500s and pD3600s, which share a similar structure. The pD2500s are compatible with one of our Leap-In transposases (LPN-1), but they do not require Marker promoter Mammalian marker Promoter 1 a transposase for good levels of expression. ORF1 signal The pD3600s are compatible with both Marker polyA of our Leap-In transposases (LPN-1 ORF1 and LPN-2), and we recommend that ORF1 poly A they only be used with a transposase. Insulator intervening sequence This is because we have deliberately attenuated the selectable markers in Leap-In Transposon the pD3600s, so multiple integration P kanamycin events are required for cell viability Promoter 2 under selection. The pD3600s are M_Kanamycin-r_* therefore capable of higher expression yields. ORF2 signal Bacterial Origin ORF2 The pD2500 and pD3600 vectors are Transposon right border ORF2 polyA constructed in a modular way that allows Insulator easy modification of component elements. Versions are available that allow expression of up to four open reading frames in addition to the selectable marker. Our stable vectors are built from several sets of mammalian functional elements: ⬤ Metabolic selectable markers ⬤ RNA processing elements Glutamine synthase (GS), dihydrofolate Introns, post-transcriptional response reductase (DHFR) elements ⬤ Drug-resistance markers ⬤ Insulators puromycin, blasticidin, hygromycin, Flanking the construct, and isolating neomycin, zeocin transcriptional units from each other ⬤ Promoters EF1a, CMV (murine and human), hybrids The total number of possible combinations of these is very large, and we do not have all of these built. However construction is modular, so it is easy for us to create custom combinations if we don’t have the combination you want. 6 www.atum.bio MAMMALIAN EXPRESSION One Vector, Two (or more) Open Reading Frames Co-transfection of separate vectors containing chain to obtain high titers. If genes encoding the genes for heavy and light chains is a common two chains are incorporated into the production method for producing antibody from transiently host genome independently, the number of transfected cells. This approach is less desirable gene copies integrated, and integration position for stable cell line generation. The production of effects, will influence the relative expression of the properly assembled antibody requires