01 Automated Protein Expression and Purification Using ExiProgen Selection Guide 267 ExiProgen EC Protein Synthesis Kit 268 ExiProgen EC-Maxi Protein

Automated Protein Expression and Purification Using ExiProgenTM Manual Protein Expression and Purification Preparation of Template DNA for Protein Expression Protein Service Protein Expression and Purification Phone: 042-930-8647 Email: proteinsupport@bioneer.co.kr

01 Automated Protein Expression and Purification Using ExiProgen Selection Guide 267 ExiProgen EC Protein Synthesis Kit 268 ExiProgen EC-Maxi Protein Synthesis Kit 270 ExiProgen EC-Tagfree Protein Synthesis Kit 271 ExiProgen EC-Disulfide Protein Synthesis Kit 272 ExiProgen His-tagged Protein Purification Kit 273 ExiProgen Cell-Based Protein Purification Kit 274

Selection Guide Overview 바이오니아에서는무세포단백질발현방법을 ExiProgen TM 에적용시킨다양한 kit 들을개발하여판매하고있습니다. 연구용효소등일반적인단백질을 ~ 100 μg 정도로합성할수있는 ExiProgen TM EC Protein Synthesis Kit, 대량으로 500 μg 내외의단백질을합성할수있는 ExiProgen TM EC-Maxi Protein Synthesis Kit, 이황화결합이 2 개이상인단백질을합성하는 ExiProgen TM EC-Disulfide Protein Synthesis Kit, His-tag 이제거된단백질을합성할수있는 ExiProgen TM EC-Tagfree Protein Synthesis Kit 을제공하고있습니다. 뿐만아니라, 재조합단백질이발현된 E. coli cell lysate 또는동물세포로부터목적단백질을정제할수있는 ExiProgen TM Histagged Protein Purification Kit, ExiProgen TM Cell-Based Protein Purification Kit 도제공하고있습니다. 원리및제품에대한보다자세한내용은 ExiProgen TM Guide Book 을참조하시기바랍니다. ExiProgen TM Guide Book 은바이오니아홈페이지 (www.bioneer.co.kr) 에서간단한신청서작성후, 무료로다운로드받으실수있습니다. www.bioneer.co.kr 267

ExiProgen EC Protein Synthesis Kit 한번에다종의단백질들의합성여부를보려고할때 Experimental Data <Detection of color> <Detection of fluorescence> sfgfp RFP AcGFP sfgfp RFP AcGFP Description 본 kit 는 ExiProgen TM 장비에서단백질을합성하는 kit 입니다. Template DNA 를첨가한 kit 를 ExiProgen TM 에장착한후, Protocol no. 902 ( 약 6 시간 ) 를가동시키면전자동으로단백질이발현되고 His-tag 를이용하여정제합니다. 1 회반응으로최대 16 종의서로다른단백질을고순도로얻을수있습니다. Features and Benefits Fully Automatic: DNA-in-Protein-out 1~10 μg DNA 를 kit 와함께 ExiProgen TM 에장착한후가동시키면전자동으로약 6 시간이내에고순도의단백질을얻을수있습니다. High Performance 세포배양으로합성이불가능한세포독성을갖는단백질의합성도가능합니다. High Purity and High Yield 각각의반응 well 당순도 90% 이상의단백질을 100 μg 내외로얻을수있습니다. Reproducibility 손쉽게 template DNA 만첨가하면원하는단백질을손쉽게얻을수있어재현성이높습니다. Application 연구용및상업용효소를포함하는단백질의연구개발에사용될수있으며, 특히다수의 mutant screening 분석에적용할수있습니다. Figure 1. Detection of fluorescence emitted from fluorescent proteins synthesized with ExiProgen TM. This result indicates that these synthesized SfGFP, RFP, and AcGFP are functionally active. Left panel: Color of each protein elution samples detected with naked eyes. Right panel: Fluorescence from protein elution samples detected with UV illuminator. 66 kda 45 kda 29 kda 20.1 kda 14.4 kda 66 kda 45 kda 29 kda 20.1 kda 14.4 kda 1 2 3 4 5 6 7 8 (well ) M E P E P E P E P E P E P E P E P 9 10 11 12 13 14 15 16 (well) M E P E P E P E P E P E P E P E P Figure 2. SDS-PAGE gel data of synthesized proteins. Each linear template DNA generated by ExiProgen TM ProXpress PCR Template Kit and those are used as template for protein synthesis with ExiProgen TM EC Protein Synthesis Kit. M: AccuLadder TM Protein Size Marker (Low)( D-2020, Bioneer) 1: SAV (13 kda), 2: RNase H (20 kda), 3: hgh (23 kda), 4: CAT (26.5 kda) 5: UDG (28 kda), 6: AcGFP (28 kda), 7: EVO (30 kda), 8: RFP (31 kda) 9: Poly A polymerase (54 kda) BIONEER 268

ExiProgen EC Protein Synthesis Kit M1 1 2 3 4 5 6 7 8 9 10 M2 66 kda 45 kda 29 kda 20.1 kda 14.4 kda 116 kda 97.4 kda 66 kda 45 kda 29 kda 20.1 kda 14.4 kda Figure 3. PAGE data of various proteins synthesized from various templates (Plasmid DNA-10~120 kda, PCR product-10~60 kda 합성가능 ). Up to 16 types of proteins can be expressed and purified simultaneously with an average of over 90% purity. Amount of loading sample is 1/35 of total sample. M1: AccuLadder TM Protein Size Marker (Low) 1: CalmL3 (17.5 kda), 2: RNase H (20 kda), 3: DUSP 3 (22 kda) 4: CAT (24 kda), 5: AcGFP (29 kda), 6: EF-Ts(34 kda) 7: VF (45 kda), 8: Poly A polymerase (50 kda) 9: M-MLV RTase (75 kda), 10: BM3 (117 kda) M2: AccuLadder TM Protein Size Marker (Broad) 66 kda 45 kda 29 kda 20.1 kda 14.4 kda M E P 300 250 200 150 100 50 0 0 10 20 30 40 50 60 70 Time(min) Base line BSA Negative control DUSP3 sample Figure 4. PAGE & enzyme activity data of DUSP3. Left panel: Expression and purification of DUSP3 (protein tyrosine phosphatase). M: AccuLadder TM Protein Size Marker (Low) E: Expression sample, P: Purification sample Right panel: DUSP3 synthesized with ExiProgen TM has an enzyme activity. DUSP3 phosphatase activity was measured by incubating with 500 μm 3-O-methylfluorescein phosphate (OMFP). (Reaction buffer: 100 mm Tris-HCl (ph 8.2), 40 mm NaCl, 1 mm DTT, 20% Glycerol) 166 kda 97.4 kda 66 kda 45 kda 29 kda 20.1 kda 14.4 kda M E P M NC1 NC2 Sample GAPDH (500 bp) Figure 5. PAGE & enzyme activity data of M-MLV RTase. Left panel: Expression and purification of M-MLV RTase. M: AccuLadder TM Protein Size Marker (Broad), E: Expression sample, P: Purification sample Light panel: Amplification of GAPDH of Human total RNA with M-MLV RTase was synthesized with ExiProgen TM. Lane M: 100 bp DNA Ladder Lane NC1: Negative control 1 (No-RNA) Lane NC2: Negative control 2 (No-enzyme) Lane Sample: 10 ng Human total RNA from HeLa cell Ordering Information Cat. no. Product Description K-7300 ExiProgen TM EC Protein Synthesis Kit, 16 rxns K-7301 ExiProgen TM EC Protein Synthesis Kit, 32 rxns K-7302 ExiProgen TM EC Protein Synthesis Kit, 96 rxns Related Product K-7400 ExiProgen TM ProXpress PCR Template Kit, 16 rxns K-2631 AccuPower ProFi Taq PCR PreMix, 96 tubes, 20 μl rxn/tube K-7350 pbivt Vectors Set-1 K-3035 AccuPrep Gel Purification Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument www.bioneer.co.kr 269

ExiProgen EC-Maxi Protein Synthesis Kit 1 mg 이상의단백질을손쉽게얻고싶을때 Features and Benefits Fully Automatic: DNA-in-Protein (in storage buffer)-out 1~6 μg DNA 를 kit 와함께 ExiProgen TM 에장착한후가동시키면전자동으로단백질을합성하고, 목적단백질정제용액을 storage buffer 로치환한상태로회수할수있습니다. Parallel Processing 1~8 종의단백질을동시에합성할수있습니다. High Purity and High Yield 각각의반응 well 당순도 90% 이상의단백질을최대 500 μg 까지얻을수있습니다. Application Description 바이오니아의특허기술인 ExiProgen TM 장비의 SECF (Stepwise Exchange Cell-Free) 단백질합성법을이용하여전자동으로많은양의단백질을합성할수있는 kit 입니다. Template DNA 를첨가한 kit 를 ExiProgen TM 에장착한후, Protocol no. 903 ( 약 25 시간 ) 을가동시키면순도 90% 이상의단백질을반응 well 당최대 500 μg 합성할수있습니다. 또한단백질발현 / 정제및투석까지모든반응이전자동으로이루어지며, 최종목적단백질은원하는 storage buffer 에용해된상태로회수할수있습니다. Experimental Data 연구용및상업용효소를포함하는단백질의연구개발에사용될수있습니다. Figure 1. Synthesis of various proteins. Proteins can be expressed and purified simultaneously with an average of over 90% purity. Amount of loading sample is 1/80 of total sample. M1: AccuLadder TM Protein Size Marker (Low) 1: CalmL3 (17.5 kda), 2: DUSP 3 (22 kda) 3: CAT (24 kda), 4: AcGFP (29 kda) 5: RFP(30 kda) 6: EF-Ts (34 kda) 7: VF (45 kda) 8: BM3 (117 kda), M2: AccuLadder TM Protein Size Marker (Broad) Ordering Information BIONEER Cat. no. Product Description K-7310 ExiProgen TM EC-Maxi Protein Synthesis Kit, 8 rxns Related Product K-7350 pbivt Vectors Set-1 K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument 270

ExiProgen EC-Tagfree Protein Synthesis Kit Histidine Tag 이제거된단백질을얻고자할때 Application 단백질의구조분석이나, 단백질의약품개발, 항원단백질합성에응용될수있습니다. Experimental Data Expression sample M 1 2 3 4 Expression sample M 1 2 3 4 66 kda 45 kda 29 kda 66 kda 45 kda 29 kda Description 바이오니아의 ExiProgen TM 장비를이용하여원형그대로의단백질을전자동으로합성할수있는 kit입니다. 목적단백질을 His-tag이있는상태로발현시킨후, 이를제거하여고순도의단백질을얻을수있는 kit입니다. Template DNA를첨가한 kit 를 ExiProgen TM 에장착한후, Protocol no. 904 ( 약 25시간 ) 를가동시키면순도 95% 이상의 His-tag이제거된목적단백질을얻을수있으며, 최종목적단백질은 storage buffer에용해된상태로회수할수있습니다. Features and Benefits Protein Synthesis without His-tag Template DNA 를 kit 와함께 ExiProgen TM 에장착한후가동시키면단백질이발현되고, 발현된목적단백질은 protease 의처리에의해서 His-tag 이제거된상태로회수됩니다. Fully Automatic: DNA-in-Protein (in storage buffer)-out 목적단백질은 storage buffer 에용해된상태로회수됩니다. Parallel Processing and High Purity 1~8 종의단백질을동시에합성할수있으며, 순도 95% 이상의단백질을얻을수있습니다. 20.1 kda 14.4 kda 20.1 kda 14.4 kda Figure 1. Expression and purification of various proteins. M: AccuLadder TM Protein Size Marker (Low) (D-2020, Bioneer) 1: DUSP3 (22 kda), 2: hgh (23 kda), 3: AcGFP (28 kda) Note) DUSP3: Dual specificity protein phosphatase 3, hgh: Human growth hormone 1. SDS-PAGE Gel Data 2. Western Blot Data (k Da) 66 45 29 20.1 M 1 2 3 2 3 Figure 2. Data of SDS-PAGE and Western blot with His-tag antibody. M: AccuLadder TM Protein Size Marker (Low) 1: Expression sample of pbivt-tev-acgfp 2: His tag-tev-acgfp (His tagged sample) 3: AcGFP (His-tag free sample) Ordering Information Cat. no. Product Description K-7320 ExiProgen TM EC-Tagfree Protein Synthesis Kit, 8 rxns Related Product K-7350 pbivt Vectors Set-1 K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument www.bioneer.co.kr 271

ExiProgen EC-Disulfide Protein Synthesis Kit Disulfide Bond 가있는목적단백질을얻고자할때 Application Pharmaceutical protein, ScFv 합성및항체스크리닝에응용될수있습니다. Experimental Data Description 바이오니아의 ExiProgen TM 장비를이용하여이황화결합 (disulfide bond) 을가지고있는목적단백질을합성하는 kit 입니다. Template DNA 를첨가한 kit 를 ExiProgen TM 에장착한후, Protocol no. 905 ( 약 36 시간 ) 를가동시키면단백질발현 / 정제및투석까지모든반응이전자동으로이루어지며, 최종목적단백질은 storage buffer 에용해된상태로회수할수있습니다. Features and Benefits Protein Synthesis with Disulfide Bond 이황화결합 (1~9 개 ) 을가지고있는단백질을합성할수있으며, 합성된단백질은고유의활성을나타냅니다. Fully Automatic: DNA-in-Protein (in storage buffer)-out 목적단백질은 storage buffer 에용해된상태로회수됩니다. Parallel Processing and High Purity 1~8 종의단백질을동시에합성할수있습니다. 66 kda 45 kda 29 kda 20.1 kda Figure 1. SDS- PAGE & enzyme activity data. Left Pannel: Expression and purification of ScFV and rpa M: AccuLadder TM Protein Size Marker (Low), 1: ScFV (it has 2 disulfide bonds.), 2: rpa (it has 9 disulfide bonds) * Amount of purification sample is 1/40 of total sample. Right panel: rpa synthesized has an enzyme activity. 1: Synthesis sample using ExiProgen TM EC-Maxi Protein Synthesis Kit 2: Synthesis sample using ExiProgen TM EC-Disulfide Protein Synthesis Kit * rpa activity was measured by incubating with chromogenic substrate S-2288. M 1 2 Amount of active rpa (ug/ml) 180 160 140 120 100 80 60 40 20 0 1 2 Ordering Information Cat. no. Product Description K-7330 ExiProgen TM EC-Disulfide Protein Synthesis Kit, 8 rxns Related Product K-7350 pbivt Vectors Set-1 K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument BIONEER 272

ExiProgen His-tagged Protein Purification Kit Cell-Free Protein 의 Purification Application His-tag 을갖는목적단백질이발현된 E.coli cell lysate 으로부터목적단백질을정제하는데사용될수있습니다. Experimental Data M CL P 45 kda 29 kda Description 바이오니아의 ExiProgen TM 을사용 (protocol no. 901, 약 2 시간 ) 하여재조합단백질이발현된 E. coli 세포파쇄액또는무세포단백질발현시료로부터 His-tag 을가지고있는목적단백질을고순도로정제하는 kit 입니다. Kit 내에는 Ni-NTA magnetic bead 와정제에필요한버퍼가 pre-filled 되어있는 cartridge 가포함되어있습니다. Ni-NTA magnetic bead 는당사가개발한자성실리카입자로외부에노출된니켈이온이단백질의 His-tag 과결합할수있어, 목적단백질을고순도로정제할수있습니다. 20.1 kda Figure 1. Purification of DUSP3 with ExiProgen TM. M: AccuLadder TM Protein Size Marker (Low) CL: Sample of recombinant E. coli cell lysate (DUSP3 expression) P: Sample of purification Features and Benefits Availability of Various Sample In vivo (E. coli 발현 ) 또는 in vitro ( 무세포발현 ) 로발현된재조합단백질의정제가가능합니다. Pre-filled Buffer Cartridge System 단백질정제에필요한모든버퍼가들어있는 pre-filled cartridge 가제공되므로, 세포파쇄액또는무세포단백질발현시료만준비하면즉시사용이가능합니다. Parallel Processing and High Purity 1~8 종의단백질을동시에합성할수있으며, 순도 90% 이상의단백질을얻을수있습니다. Ordering Information Cat. no. Product Description K-7220 ExiProgen TM His-Tagged Protein Purification Kit, 16 rxns Related Product D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument www.bioneer.co.kr 273

ExiProgen Cell-Based Protein Purification Kit Modified Protein 의 Purification Application His-tag 을갖는목적단백질이발현된동물세포또는세포배양액으로부터목적단백질을정제하는데사용될수있습니다. Experimental Data Description 바이오니아의 ExiProgen TM 을사용 (protocol no. 906, 약 7 시간 ) 하여 eukaryotic cell 에서발현된 His-tag 을가지고있는목적단백질을고순도로정제하는 kit 입니다. 본 kit 에는 cell lysis buffer, Ni-NTA magnetic bead, 정제및 storage buffer 등이포함되어있어, 목적단백질이포함된세포추출물또는세포를첨가하면전자동으로목적단백질을고순도로정제한후, storage buffer 에용해된상태로회수합니다. Figure 1. Purification of AcGFP with ExiProgen TM. M: AccuLadder TM Protein Size Marker (Low) CL: Sample of recombinant HEK293 cell (AcGFP expression) P: Sample of purification Features and Benefits Fully Automatic: Cell-in-Protein (in storage buffer)-out 재조합단백질 (His-tagged protein) 발현세포를준비하여 kit 에넣은후 ExiProgen TM 에장착한후가동시키면, storage buffer 에용해된목적단백질을회수할수있습니다. Pre-filled Buffer Cartridge System 단백질정제에필요한모든버퍼가들어있는 pre-filled cartridge 가제공되므로, 발현시료만준비하면즉시사용이가능합니다. Parallel Processing and High Purity 1~8 종의단백질을동시에합성할수있으며, 고순도의단백질을얻을수있습니다. Ordering Information Related Product Cat. no. Product Description K-7230 ExiProgen TM Cell-Based Protein Purification Kit, 16 rxns D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl A-5041 ExiProgen TM, Instrument BIONEER 274

02 Manual Protein Expression and Purification Selection Guide 276 AccuRapid Protein Synthesis Kit 277 AccuRapid Cell-Free Protein Expression Kit 278 MagListo His-tagged Protein Purification Kit 280

Selection Guide Overview 본제품군은매뉴얼타입의제품군으로, 용도에따라사용자가선택할수있도록단백질발현에서정제까지수행할수있는제품 (AccuRapid TM Protein Synthesis Kit) 과, 단백질발현만수행하는제품 (AccuRapid TM Cell-Free/Midi/Maxi Protein Expression Kit), 단백질정제만수행하는제품 (MagListo TM His-tagged Protein Purification Kit) 으로나뉘어져있습니다. 원리및제품에대한보다자세한내용은 ExiProgen TM Guide Book 을참조하시기바랍니다. ExiProgen TM Guide Book 은바이오니아홈페이지 (www.bioneer.co.kr) 에서간단한신청서작성후, 무료로다운로드받으실수있습니다. BIONEER 276

AccuRapid Protein Synthesis Kit The Easy Method for Protein Synthesis!! Application 연구용및상업용효소를포함하는단백질의연구개발에사용될수있습니다. Experimental Data Expression sample Purification sample Description AccuRapid TM Protein Synthesis Kit 는매뉴얼방식으로원하는단백질의발현을확인할수있는 kit 입니다. 본 kit 는 E. coli 무세포발현및 Ni-affinity 정제실험에사용되는모든구성품을포함하고있으며, 반응당정제된목적단백질을 100 μl 내외로회수할수있습니다. Figure 1. SDS-PAGE data of various proteins synthesized from various templates. M: AccuLadder TM Protein Size Marker (Broad) 1: CalmL3 (17.5 kda), 2: DUSP 3 (22 kda), 3: CAT (24 kda), 4: AcGFP (29 kda) 5: ERFP (31 kda), 6: EF-Ts (34 kda), 7: VF (45 kda), 8: BM3 (117 kda) Features and Benefits High Simplicity and High Performance 세포를이용하지않고시험관튜브내에서손쉽게단백질을합성할수있으며, 기존의세포배양방법으로발현이불가능했던단백질도합성이가능합니다. Flexibility T7 발현시스템에서단백질발현이가능한다양한 template DNA 로부터원하는단백질의발현및정제여부를손쉽게확인할수있습니다. Compatibility ExiProgen TM EC Protein Synthesis Kit 와호환이가능합니다. Ordering Information Cat. no. Product Description K-7280 AccuRapid TM Protein Synthesis Kit, 5 rxns Related Product K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns K-3035 AccuPrep Gel Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl K-7350 pbivt Vectors Set-1 K-7400 ExiProgen TM ProXpress PCR Template Kit, 16 rxns www.bioneer.co.kr 277

AccuRapid Cell-Free Protein Expression Kit Screening for Protein Expression Experimental Data 1. AccuRapid TM Protein Expression Kits 를이용한 AcGFP 단백질 (Positive Control) 발현 Description 본 kit 는반응튜브내에서 template DNA 를이용하여 3 시간만에원하는단백질의발현을확인할수있는 kit 로서 E. coli extract 와 master mix 를포함하고있습니다. 이중 E. coli extract 에는 T7 RNA polymerase, ribosome 등이포함되어있고, master Mix 는아미노산, rntps, 에너지원등이포함되어 template DNA 만있으면, 손쉽게목적단백질의발현을확인할수있습니다. Features and Benefits High Speed 3 시간만에 DNA 로부터단백질을발현할수있습니다. Flexibility T7 발현시스템에서단백질발현이가능한다양한 template DNA 로부터원하는단백질의발현여부를손쉽게확인할수있습니다. High Performance Host 세포에독성을나타내어기존의 cloning 방법으로발현이불가능했던단백질의발현이가능합니다 Compatibility 본 kit 는 ExiProgen TM 단백질합성 kit 류와호환이가능하며, template DNA 의최적농도를찾는데사용할수있습니다. Application 연구용및상업용효소를포함하는단백질의연구개발에사용될수있습니다. Figure 1. Positive control protein expression data of AccuRapid TM Protein Expression Kits. (SDS-PAGE and Coomassie Brilliant Blue staining) M : AccuLadder TM Protein Size Marker (Low) (Bioneer Cat.no. D-2020), 1,3,5 : Negative control(no-dna), 2,4,6 : Positive control (pbivt-acgfp) Note) K-7250-45 μl rxn, K-7260-1 ml rxn, K-7270-10 ml rxn 2. 다양한단백질의발현 66 kda 45 kda 29 kda 20.1 kda 14.4 kda M 1 2 3 4 5 6 PTP18 GFP CAT Figure 2. Various protein expression data (SDS-PAGE and Coomassie Brilliant Blue staining) M : AccuLadder TM Protein Size Marker (Low) (Bioneer Cat. no. D-2020) 1,3,5: Negative control(no-dna), 2: CAT (Chloramphenicol acetyl transferase), 4: GFP (Green fluorescence protein) 6: PTP1B(Protein tyrosine phosphatase 1B) BIONEER 278

AccuRapid Cell-Free Protein Expression Kit 3. Template DNA 의최적 DNA 농도 screening Figure 3. Determination of the optimal amount of template DNA for the expression of pbivt-gfp. (SDS-PAGE and Coomassie Brilliant Blue staining) M : AccuLadder TM Protein Size Marker (Low) (Bioneer Cat. no. D-2020) 1: Negative Control(No-DNA), 2 : CAT (Chloramphenicol acetyl transferase),3 ~9: pbivt-gfp(50, 100, 200, 400, 600, 800(ng)) Ordering Information Cat. no. Product Description K-7250 AccuRapid TM Cell-Free Protein Expression Kit, 45 μl x 24 rxns K-7260 AccuRapid TM Midi Protein Expression kit, 1 ml x 5 rxns K-7270 AccuRapid TM Maxi Protein Expression kit, 10 ml x 1 rxn Related Product K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns K-3035 AccuPrep Gel Purification Kit, 200 rxns D-1030 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500 μl K-7350 pbivt Vectors Set-1 K-7400 ExiProgen TM ProXpress PCR Template Kit, 16 rxns www.bioneer.co.kr 279

MagListo His-tagged Protein Purification Kit Fast Micro Scale Purification of 6x His-tagged Protein Experimental Data Description Ni-NTA magnetic silica resin 은 6x Histidine 으로 cloning 된단백질과결합할수있는 Ni 2+ 를지니고있어, 실험실에서단백질발현을 screening 하는데필요한 native 한조건에서실험할수있도록최적화되어있습니다. Ni-NTA magnetic silica resins 는 20% EtOH 1 ml 안에 10% 의부피를차지하고있으면, resin 1 ml 을사용하여 6x Histidine tagged protein 를약 500 μg 정도 binding 할수있습니다. Figure 1. Purification of 6x His-tagged protein. M: Protein broad marker (Bioneer, D-2010) L: Loading sample 1: Unbound sample 2: Eluted sample (MagListo TM His-tagged Protein Purification Kit) Features and Benefits High Speed Ni-NTA Magnetic bead 를이용한 wide range 의 His-tagged proteins 를빠르고쉽게정제 High Performance SSMB(Spherical Shape Magnentic Beads) 에의한고순도 protein 획득 High Efficiency Crude cell lysate 를이용한 screening 공정에서의높은효율성 Application 본 kit 는매뉴얼방식으로 His-tag 을갖는목적단백질을정제하는데사용될수있습니다. Figure 2. Protein size effect on purification. M : Protein broad marker (Bioneer, D-2010) 1 : Size 94 kda His-tagged protein 2 : Size 67 kda His-tagged protein 3 : Size 38 kda His-tagged protein 4 : Size 27 kda His-tagged protein 5 : Size 25 kda His-tagged protein Ordering Information Cat. no. Product Description K-7200 MagListo TM His-tagged Protein Purification Kit Related Product D-2020 AccuLadder TM Protein Size Marker (Low), 500 μl D-2010 AccuLadder TM Protein Size Marker (Broad), 500μl BIONEER 280

03 Preparation of Template DNA for Protein Expression Preparation of Template DNA for Protein Expression 282 ExiProgen ProXpress PCR Template Kit 283 pbivt Vector Set-1 285

Preparation of Template DNA for Protein Expression Overview 목적단백질을발현하기위해서는무세포단백질발현용 template DNA 를먼저준비해야합니다. Template DNA 는 T7 promoter - Ribosome Binding Site (RBS) - Target gene (6x His-tag sequence 포함 ) - T7 terminator 의구조를가지고있어야하며, 그구조는아래의그림과같습니다. 또한 target gene 은 start codon 과 stop codon 을가지고있어야합니다. 또한선택적으로 Ni-affinity 정제를하기위하여 5 말단또는 3 말단에 6x His-tag 을가지고있어야합니다. 발현벡터는바이오니아에서판매하는 in vitro translation 전용벡터인 pbivt 벡터 (Cat. no. K-7350) 에 cloning 하여사용할수있고, 그밖에도 pk7, pivex, pet 벡터등을사용할수있습니다. PCR product 로부터 cloning 을하지않고바로 template DNA 를제작하려면당사의 ExiProgen TM ProXpress PCR Template Kit (Cat. no. K-7400, 7401) 를이용하여제작할수있습니다. 많은종류의단백질을소량으로빠르게만들어서성능을평가해볼때매우유용한 kit 로서 genomic DNA, cdna 또는 T-vector 등에서간편하게 template DNA 제작부터단백질합성까지 1 일내로진행할수있는장점이있습니다. 바이오니아에서는 template DNA 를 in vitro translation vector 에클로닝된상태로제공받으실수있는 Gene Synthesis Service 도제공하고있습니다. 자세한내용은홈페이지 (www.bioneer.co.kr) 를참조하시기바랍니다. 1) NH constructs (5 end 6x His-tagged template) T7 Promoter---RBS ATG 6x His-tag -----------------------------------stop T7 terminator 2) CH constructs (3 end 6x His-tagged template) T7 Promoter---RBS ATG --------------------------------- 6x His-tag - stop T7 terminator BIONEER 282

ExiProgen ProXpress PCR Template Kit Fast and Parallel Template DNA Preparation with PCR Description Cloning 과정없이 PCR 반응을이용하여무세포단백질발현 (cellfree protein expression) 용 template DNA 를제작할수있는 kit 로, 표적유전자증폭용 1 차 primer set 를제외한 template DNA 제조반응에필요한모든구성품 (2 차 primer set, PCR premix 등 ) 이 kit 에포함되어있습니다. 본 kit 로제작된단백질발현용 template DNA 는당사의무세포단백질발현및합성제품군에사용가능합니다. Features and Benefits Rapid & Easy Preparation of Linear Template DNA (for Protein Expression) 목적단백질의유전자를포함하는 cdna, genomic DNA 등으로부터두단계의 PCR 반응을통해단백질합성용 template DNA 를제조할수있으며, ExiProgen TM kit 와연계하여하루만에목적단백질합성을완료할수있습니다. Accurate Protein Template by High Fidelity PCR 본 kit 는당사의대표적인 long & high fidelity PCR Premix 인 AccuPower ProFi Taq PCR Premix 를사용하여, PCR 반응시생길수있는 error rate 를줄여주기때문에정확한 sequence 를가진단백질발현용 template DNA 를제작할수있습니다. High Efficiency 본 kit 를이용하여제조한단백질발현용 template 는 ExiProgen TM 에최적화되어소량의 DNA 만으로도 100 μg 이내의단백질을합성할수있습니다. (ex: template DNA 길이에따른최대단백질합성을위한권장 DNA 사용량 : 1 kb 이하 - 500 ng, 1 ~ 2 kb - 1 μg) Application 무세포단백질발현용 template DNA 제작에사용될수있습니다. Experimental Data st 1 PCR products M1 1 2 3 4 5 6 7 8 9 st 2 PCR products M1 1 2 3 4 5 6 7 8 9 M2 Figure 1. Agarose gel data of template DNA synthesized by each PCR. Synthesis of linear template DNA with ExiProgen TM ProXpress PCR Template Kit. M1: 100 bp DNA Ladder (D-1030, Bioneer), M2: 1 kb DNA Ladder (D-1040, Bioneer) 1: SAV (Template - pt), 2: RNase H (Template - BL21 (DE3) gdna), 3: hgh (Template - pt) 4: CAT (Template - pbivt), 5: UDG (Template - BL21 (DE3) gdna), 6: AcGFP (Template - pbivt) 7: EVO (Template - pt), 8: RFP (Template - pivex), 9: Poly A polymerase (Template - pet15b) www.bioneer.co.kr 283

ExiProgen ProXpress PCR Template Kit Figure 2. SDS-PAGE gel data of synthesized proteins. Each linear template DNA generated by ExiProgen TM ProXpress PCR Template Kit and those are used as template for protein synthesis with ExiProgen TM EC Protein Synthesis Kit. M: AccuLadder TM Protein Size Marker (Low)( D-2020, Bioneer) 1: SAV (13 kda), 2: RNase H (20 kda), 3: hgh (23 kda), 4: CAT (26.5 kda), 5: UDG (28 kda) 6: AcGFP (28 kda), 7: EVO (30 kda), 8: RFP (31 kda), 9: Poly A polymerase (54 kda) Ordering Information Cat. no. Product Description K-7400 ExiProgen TM ProXpress PCR Template Kit, 16 rxns K-7401 ExiProgen TM ProXpress PCR Template kit, 32 rxns Related Product N-8229 1차 primer F/R sets (N-terminus 6x His-tag), each 5 nmole 1 N-8230 1차 primer F/R sets (C-terminus 6x His-tag), each 5 nmole 1 K-2631 AccuPower ProFi Taq PCR PreMix, 96 tubes, 20 μl rxn/tube K-3034 AccuPrep PCR Purification Kit, 200 rxns K-3035 AccuPrep Gel Purification Kit, 200 rxns D-1030 AccuLadder TM 100 bp DNA Ladder, 250 μl (135 ng/μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) 1 Oligo synthesis service 문의 BIONEER 284

pbivt Vector Set-1 바이오니아무세포단백질합성전용벡터 Description 바이오니아의무세포단백질합성전용벡터인 pbivt 벡터세트 (pbivt-1, pbivt-2) 는당사의무세포단백질발현및합성의모든 kit 에적용가능합니다. 본벡터세트는 N-terminal (pbivt-1) 또는 C-terminal (pbivt-2) 에 6x His-tagging 이가능하며, 구조는아래와같습니다. Application 무세포단백질발현용 template DNA 제작에사용될수있습니다. Ordering Information Cat. no. Product Description K-7350 pbivt Vector Set-1 Related Product K-3030 AccuPrep Plasmid Extraction Kit, 200 rxns K-3034 AccuPrep PCR Purification Kit, 200 rxns D-1030 AccuLadder TM 100 bp DNA Ladder, 250 μl (135 ng/ μl) D-1040 1 kb DNA Ladder, 500 μl (130 ng/μl) E-3061 T4 DNA Ligase, 20,000 U www.bioneer.co.kr 285

04 Protein Service Protein Synthesis Service (with ExiProgen ) 287 Protein Expression Service (with ExiProgen ) 289

Protein Synthesis Service (with ExiProgen ) Description 바이오니아의 Protein Synthesis Service 는 ExiProgen TM 단백질합성시스템을이용하여, 고객의 DNA 로부터목적단백질을합성하여제공해드리는서비스입니다. 서비스내용 Protein Synthesis Service 는당사의전자동단백질합성장비 ExiProgen TM 으로단백질을합성해드리는서비스로, E. coli 무세포발현시스템은 T7 발현시스템을기반으로하여단백질을발현하며, 발현된단백질은 6x His-tag affinity 를이용하여정제한후, 고객이원하는 storage buffer 로 dialysis 하여제공해드리는고객맞춤형서비스입니다. Saving Time with Bioneer s protein synthesis service In vitro translation 전용벡터또는 pet 계열의 E. coli 발현벡터를가지고계신경우, 빠른결과를얻으실수있습니다. * Eukaryotic 계열의유전자는 E. coli 에코돈최적화가이루어졌을경우, 단백질합성에유리합니다. www.bioneer.co.kr 287

Protein Synthesis Service (with ExiProgen ) 서비스정보 시료유형시료제공량 DNA size 서비스진행방식 시료유형및제공량 1) 1. E. coli expression vectors 1 E. coli 무세포발현벡터 (pbivt, pivex 등 ) 2 pet 등의 E. coli 발현벡터 2. Non expression vectors 1 Cloning vector 2 Target gene PCR product 10 μg 이상 2 μg 이상 < 3.5 kb (Insert 기준 ) < 1.5 kb (Target gene 기준 ) 1 주문접수및견적서발송 2 시료수거 3 시료확인 ( 농도및순도확인 ) 4 DNA 농도별단백질발현확인 5 ExiProgen TM 으로단백질합성 6 단백질시료발송및비용청구 1 주문접수및견적서발송 2 시료수거 3 Target 유전자증폭을위한 Primer 합성 4 단백질발현구조를갖는 PCR product 제작 5 ExiProgen TM 으로단백질합성 6 단백질시료발송및비용청구 기본비용 600,000 원 (Molecular weight 30 kda 이하 ) 서비스비용 단백질크기 추가비용 Molecular weight 31 kda ~ 80 kda 100,000 원 /10 kda 추가 Molecular weight >81 kda 1,000,000 원추가 합성량 2) 기본비용 0.1 mg 추가비용없음 추가비용 0.2 mg ~ 1 mg 100,000 원 /0.1 mg 추가 단, 단백질합성이되지않을시, Protein Expression Service 로자동전환되며, Data report 발송과함께 set up charge 100,000 원이청구됩니다. Storage buffer 50 mm Tris-Cl(pH 7.6), 100 mm NaCl, 1 mm DTT, 0.1 mm EDTA, 0.05% NaN 3, 50% Glycerol 단백질농도는 0.1~1 mg/ml 로제공되며, 원하시는 storage buffer 의조성이있는경우 order sheet 에기록하여주시기바랍니다. 단, 위의조성이외에추가로첨가되는고가의시약이있는경우, DNA 시료제공시함께제공해주셔야서비스가가능합니다. 소요기간 (Working day 기준 ) 5~14 일이내 ( 소요기간은기본합성량 100 μg 을기준으로합니다. 단, 시료의순도가낮거나 DNA 증량이필요한경우소요기간은증가할수있으며, 이경우는미리고지해드립니다. ) 1) Plasmid DNA 의양은최소 10 μg 이상, 순도 A260/280 : 1.7~2.0, A260/230 : >1.5 이상이어야하며, 이조건에부합되지않는경우에는 DNA 증량및정제서비스가추가되어, 추가비용이발생할수있습니다. 2) 합성량은목적단백질의순도와관계없이, 전체시료의양의의미합니다. * 기타서비스와관련된문의사항은 proteinsupport@bioneer.co.kr 메일또는단백질연구팀 (042-930-8647) 으로문의바랍니다. BIONEER 288

Protein Expression Service (with ExiProgen ) Description 바이오니아의 Protein Expression Service 는고객의유전자가당사의 ExiProgen TM 으로단백질합성이가능한지여부를확인해드리는서비스입니다. 서비스내용 Protein Expression Service 는당사의 E. coli 무세포단백질발현시스템에서고객의 DNA 가단백질발현이가능한지여부를확인해드리며, 또한해당 DNA 로당사의전자동단백질합성장비 ExiProgen TM 에서단백질합성 ( 발현및정제포함 ) 이가능한지를동시에확인해드리는서비스입니다. 본서비스에이용되는 E. coli 무세포발현시스템은 T7 발현시스템을기반으로하여단백질을발현하며, 발현된단백질은 6x His-tag affinity 를이용하여정제합니다. 서비스정보 서비스의결과는 data report 를통해제공되며, 자세한제품정보는하기와같습니다. 시료유형시료제공량 DNA size 서비스진행방식 시료유형및제공량 1) 1. E. coli expression vectors 1 E. coli 무세포발현벡터 (pbivt, pivex 등 ) 2 pet 등의 E. coli 발현벡터 2. Non expression vectors 1 Cloning vector 2 Target gene PCR product 10 μg 이상 2 μg 이상 < 3.5 kb (Insert 기준 ) < 1.5 kb (Target gene 기준 ) 1 주문접수및견적서발송 2 시료수거 3 시료확인 ( 농도및순도확인 ) 4 DNA 농도별단백질발현확인 5 ExiProgen TM 으로단백질합성확인 6 Data report 발송및비용청구 1 주문접수및견적서발송 2 시료수거 3 Target 유전자증폭을위한 Primer 합성 4 단백질발현구조를갖는 PCR product 제작 5 ExiProgen TM 으로단백질합성확인 6 Data report 발송및비용청구 서비스비용 Expression vectors Non expression vectors 시료당 100,000 원 시료당 200,000 원 소요기간 (Working day 기준 ) Expression vectors Non expression vectors 5 일이내 ( 단, 시료의순도가낮거나 DNA 증량이필요한경우소요기간은증가하며, 이경우는미리고지해드립니다. ) 14 일이내 1) Plasmid DNA 의양은최소 10 μg 이상, 순도 A260/280: 1.7~2.0, A260/230: >1.5 이상이어야하며, 이조건에부합되지않는경우에는 DNA 증량및정제서비스가추가되어, 추가비용이발생할수있습니다. * 기타서비스와관련된문의사항은 proteinsupport@bioneer.co.kr 메일또는단백질연구팀 (042)930-8647) 으로문의하여주시기바랍니다. www.bioneer.co.kr 289

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