김도희_HT protein expression 표준 보고서_v1.5

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은영 구
6 years ago
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1 Production of Recombinant Proteins For Further Information, Please Contact Neurogenex Co., Ltd. Biotechnology Incubating Center "Golden Helix" Seoul Natl' Univ. Seoul , Korea TEL FAX

Generation of Recombinant Baculovirus Gene of Interest Construction of Maste Clone KmR PCR Site-specific Recombination Sequencing Construction of Master Clone Site-specific Recombination Production

2 Generation of Recombinant Baculovirus Gene of Interest Construction of Maste Clone KmR PCR Site-specific Recombination Sequencing Construction of Master Clone Site-specific Recombination Production of Baculovirus GOI Tag GOI Poly A Baculovirus DNA (130 kb) Baculovirus Production Purification Screening Plaque Isolation Large-Scale Amplification Production of Baculovirus Protein Purification Recombinant Baculovirus

3 High Throughput Cloning CDS (bp) PK0011 PK0012 PK0029 PK0030 PK0046 PK0051 PK0063 PK0068 PK0097 PK0098 TF0006 TF0008 TF0019 TF0021 TF0039 TF PK0014 PK0015 PK0031 PK0032 PK0052 PK0053 PK0069 PK0070 PK0099 PK0100 TF0009 TF0010 TF0025 TF00 27 TF0044 TF0046 GFP Gus PK0016 PK0018 PK0033 PK0034 PK0054 PK0055 PK0071 PK0072 PK0101 PK0104 TF0011 TF0012 TF0028 TF0029 PK0003 PK0004 PK0019 PK0020 PK0037 PK0038 PK0056 PK0057 PK0075 PK0080 PK0105 PK0106 TF0013 TF0014 TF0031 TF PK0005 PK0006 PK0021 PK0024 PK0039 PK0042 PK0058 PK0060 PK0083 PK0084 KI0001 KI0002 TF0015 TF0016 TF0034 TF PK0007 PK0010 PK0027 PK0028 PK0043 PK0044 PK0061 PK0062 PK0085 PK0093 TF0002 TF0004 TF0017 TF0018 TF0036 TF PCR amplification with gene-specific primer and adaptor primer were carried out on 94 clones for high throughput cloning. Gene Size = CDS + 64 bp

Recombinant Baculovirus CDS (bp) PK0011 PK0012 PK0029 PK0030 PK0046 PK0051 PK0063 PK0068 PK0097 PK0098 TF0006 TF0008 TF0019 TF0021 TF0039 TF0043 582 573 1209 1308 966 1953 1644 1047 876 237 1005 1092

PK0101 PK0104 TF0011 TF0012 TF0028 TF0029 PK0003 PK0004 PK0019 PK0020 PK0037 PK0038 PK0056 PK0057 PK0075 PK0080 PK0105 PK0106 TF0013 TF0014 TF0031 TF0032 717 1809 1188 1080 1938 1245 1221 645 1245

PK0021 PK0024 PK0039 PK0042 PK0058 PK0060 PK0083 PK0084 KI0001 KI0002 TF0015 TF0016 TF0034 TF0035 2784 237 1032 993 831 1809 576 1353 738 1563 492 594 2760 747 1311 1395 PK0007 PK0010 PK0027 PK0028

4 Recombinant Baculovirus CDS (bp) PK0011 PK0012 PK0029 PK0030 PK0046 PK0051 PK0063 PK0068 PK0097 PK0098 TF0006 TF0008 TF0019 TF0021 TF0039 TF PK0014 PK0015 PK0031 PK0032 PK0052 PK0053 PK0069 PK0070 PK0099 PK0100 TF0009 TF0010 TF0025 TF00 27 TF0044 TF0046 GFP Gus PK0016 PK0018 PK0033 PK0034 PK0054 PK0055 PK0071 PK0072 PK0101 PK0104 TF0011 TF0012 TF0028 TF0029 PK0003 PK0004 PK0019 PK0020 PK0037 PK0038 PK0056 PK0057 PK0075 PK0080 PK0105 PK0106 TF0013 TF0014 TF0031 TF PK0005 PK0006 PK0021 PK0024 PK0039 PK0042 PK0058 PK0060 PK0083 PK0084 KI0001 KI0002 TF0015 TF0016 TF0034 TF PK0007 PK0010 PK0027 PK0028 PK0043 PK0044 PK0061 PK0062 PK0085 PK0093 TF0002 TF0004 TF0017 TF0018 TF0036 TF Recombinant baculoviruses were produced in a 96-well plate. Inserted genes were analyzed by PCR amplification Gene Size = CDS bp Used primer for PCR identification Forward: 5 -ACC ATC TCG CAA ATA AAT AAG-3 Reverse: 5 -CAA CAA CGC ACA GAA TCT AGC-3

5 HTS Screening Expression and Solubility Screening: Purification Screening: Culture scale: 96-well plate Proteins were resolved on 12% SDS-PAGE. Anti-GST-HRP (1 : 5,000 Santacruz. Inc) was used for western detection. The red box indicates selected clone for large scale culture and purification.

Optimization of Protein Expression Plaque Isolation: M N 1 2 3 4 5 6 M N 1 2 3 4 5 6 98 64 50 36 22 M: SeeBlue_Plus2

N 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 M: SeeBlue_Plus2 (pre-stained standard, Invitrogen) N: Negative control 1: 24 h p.i. 2: 36 h p.

6 Optimization of Protein Expression Plaque Isolation: M N M N M: SeeBlue_Plus2 (pre-stained standard, Invitrogen) N: Negative control 1 ~ 6: Isolated plaque #1 ~ #6 The red digit indicates selected clone for large scale culture. Optimization of Protein Expression: Total Insoluble Soluble M N Total Insoluble Soluble M N M: SeeBlue_Plus2 (pre-stained standard, Invitrogen) N: Negative control 1: 24 h p.i. 2: 36 h p.i. 3: 48 h p.i. 4: 60 h p.i. 5: 72 h p.i. Anti-GST-HRP (1 : 10,000 Santacruz. Inc) was used for western detection. The red digit indicates selected optimal time for protein expression. *p.i.: post infection

Purification M T I S Ft W P Elution buffer Purified wash M: SeeBlue_Plus2 (pre-stained standard, Invitrogen) T: Total protein (48 h p.i.) I: Insoluble protein S: Soluble protein (loading) Ft: Flow-through W: Washing P: Purified protein One microgram of purified protein was loaded on 12% SDS-PAGE.

7 Purification M T I S Ft W P Elution buffer Purified wash M: SeeBlue_Plus2 (pre-stained standard, Invitrogen) T: Total protein (48 h p.i.) I: Insoluble protein S: Soluble protein (loading) Ft: Flow-through W: Washing P: Purified protein One microgram of purified protein was loaded on 12% SDS-PAGE. Product Description: N-terminal HisGST-fused recombinant protein. Estimated Size: 50.5 kda Formulation: 100 recombinant protein in 100 in PBS with 20% glycerol

8 Data Summary Data summary baculovirus 3. (Plaque isolation) Completion Gene Baculovirus ID Clone ID Volume Completion - NBS NGX- PK /vial Virus Protein # of vials Titer Volume # of vials Concentration Storage conditions 1 vial > 10 8 PFU/ 100 /vial 5 vials 1.0 /(100 /vial) PBS with 20% glycerol *PFU: Plaque Forming Unit

9 User Guide 27 o C. Sf21 Spnnier bottle suspension Culture.

10 User Guide 5. (6-well ) 1) Cell counting (working cell 5X10 5 /ml), 5.0 X 10 5 cells/m (Sf900II-SFM + 5%FBS). 2) well2 mlx 6 well (1X10 6 / well). 3) 30 well plate, old media. 4) Baculovirus 200 ul (10 MOI, Baculovirus titer = 1X10 8 pfu/ml) well. 5) 30~1 27 o C. (10 ) 6) Sf900II-SFM + 5%FBS 2 ml 27 o C 2~3. 7) Baculovirus media. 1.5 ml (7.5 X 10 5 cells). 4 o C. 8) PBS 500 ul. 9) PBS lysis buffer 200 ul6 X SDS loading buffer 40 ul. (Optional : ) 10) 95 o C 5, rpm 1. 11) 15 ul. (SDS-PAGE or Western blot) * 100 mm, 150 mm, Roller bottle 6-well. Virus titer (1X10 8 pfu/ml)* *Baculovirus titer1 x 10^8 pfu/ml, 10 MOI.

11 AdHTS High Throughput Recombination Adenovirus Production System Adenovirus, the proven gene delivery system AdHTS, your partner of functional genomics

BacHTS High Throughput Recombinant Baculovirus Production System 203 101 69 44 H-CDK1 H-CDK2 CDK4 H-CDK6 H-CAK H-PKCa H-MEK1 H-ERK1 H-ERK2 H-AKTDD H-ILK

12 BacHTS High Throughput Recombinant Baculovirus Production System H-CDK1 H-CDK2 CDK4 H-CDK6 H-CAK H-PKCa H-MEK1 H-ERK1 H-ERK2 H-AKTDD H-ILK H-TGFb RI H-TGFb RII GST -Abl GST -p16 cdc37 LacZ Polyhedrin Baculovirus expressing 96 Yeast Kinases (GFP-fused) were generated using BacHTS from 96-well

13 Limited Product Warranty For Research Use Only. Not for Diagnostic or Therapeutic Use. Neurogenenx shall have no liability for any direct, indirect, consequential or incidental damages arising from the use, the results of the use or the inability to use any product. Neurogenex shall be not responsible for failure or delay in performance of its obligations related to the service due to causes beyond its reasonable control, including but not limited to governmental actions, fire, labor difficulty, shortages, civil disturbances, transportation problems, interruptions of power or communications, or natural disasters. The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. Neurogenex will not be held responsible for patent infringements or other violations that may occur with the use of our product. Neurogenex is a patent-pending related to high throughput system and method for producing recombinant virus using site-specific recombination, BacHTS. Neurogenex is a patent-pending related to a method for producing recombinant viruses using site-specific recombination, AdHTS. This recombinant baculovirus expression system is the subject of one or more of US patents 4,745,051; 4,879,236; 5,155,037; and 5,278,050. Use of BP and LR recombination described in Gateway TM System is the subject of one or more of US patent 5,888,732; 6,143,557; 6,171,861; 6,270,969; and 6,277,608 and/or other pending U.S. and foreign patent applications owned by Invitrogen Corporation. Use of the CMV promoter is covered under U.S. Patent No. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation. 6xHis-Taq is under a separate patent owned by Hoffmann-La Roche (No. 4,889,818) that Promega is also challenging. The GST tag technology is covered under U.S. Patent No. 5,654, 176 issued to Amrad Corporation Limited. Mutant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a license from Aurora Biosciences Corporation under U.S. Patent No. 5,625,048, 5,777,049, 6,054,321, and 5,804,387. Flag and Anti-FLAG are registered trademarks of Sigma-Aldrich Co.

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