Scaling-up E. coli fermentation, E. coli soluble protein production, and inclusion body refolding expertise from research to GMP production.
Protein Sciences: Early-Stage R&D E. coli Platform
Complex Protein Expertise The production and analytical assessment of proteins produced from E. coli can be challenging and complex. At WuXi Biologics, our expert team of highly trained scientists can perform a wide array of protein research activities and analyses from in vivo biotinylation, major histocompatibility complex (MHC) studies, cytokine production and screening and protein refolding. Our team has extensive experience with difficult proteins and routinely conducts projects requiring protein expression optimization, evaluation and use of a wide variety of labels, tags, and chaperones, and the fermentation and co-expression of proteins.
E. coli Platform Features:
Projects start from customer-provided amino acid sequence (with codon optimization), DNA sequence, or plasmids Use of multiple purification steps to deliver high quality product A toolbox for hard-to-express targets, including host strains, culture conditions, and plasmid designs High throughput screening for evaluation of protein refolding conditions (over 200 refolding conditions established) Cell-free expression system available for the screening of hard-to-express proteins and various mutants Protein expression condition optimization Flexible product purification and analytical QC packages
Experience with Difficult-to-Express Proteins
Intracellular Expression: • Soluble • Inclusion body Secreted Expression: • Periplasm Different Strains (K/B) and Hosts Available: • Protease deficiency
Culture Condition
Host Strain
Plasmid Design
• Disulfide bond enhancer • Minimal leaky expression • Low-rate producer • Low temperature expression • Low endotoxin Shake Flask Studies: • 0.1-10 L various parameters evaluated: Temperature, inducer, cell density, culture time, media, etc. HTP Refolding Platform Chaperones: • HSP: DNaK, DNaJ, GrpE, GroEL/ES Fusion Tag and Removal: • His/Flag/GST Target Protein Engineering
Gene Synthesis 1-2 weeks
Shake Flask Expression <1 week
Purification and QC 1 week
E-coli Large-Scale Fermentation
Service Scope
Advantage
Plasmid Construction
Various Promoter Systems
Shake Flask Optimization
Different Feed Control: DO/pH-stat, Exponential
• MBP/NusA: solubility, folding • SUMO/Ubiquitin: solubility Signal Peptide: • Sec/Tat pathway Vector, Promoter, Copy Number, and more
1-5 L Scale up Up to 50 L
Higher Titer (1-20 g/L) Short Culture Period (1-5 days)
Package Selection Guide
Our various E. coli protein production packages can start with an optional protein expression optimization study. We offer multiple scales of production up to 50 L, purification of both the soluable protein and inclusion body fractions and flexible QC analysis.
Popular packages
Features
Timeline
QC and Deliverable
Expression optimization
4-8 conditions
1 week
Expression report, soluble vs. insoluble fractions
Refolding screening
96 DWP, >200 conditions to choose
2-3 weeks
Refolding report
5 L, 10 L and 50 L (fermentation) Additional QC 1 L (shake flasks)
Fed batch RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, cIEF, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing Tag removal, biotinylation, endotoxin removal Concentration, Caliper (R/NR) or SDS-PAGE, LC/MS 4 weeks 3-4 weeks
Case Study: E. coli Expression Optimization
In this case study, we performed an expression optimization study to improve protein expression.
Key Challenge: Low expression
Expression condition optimization
Parameters
Original
Optimized
Medium Strain
Platform Medium BL21 (DE3) 20 1-5 24
Optimized Medium BL21 star (DE3) 37 >100 24
Expression Analysis
Final Products M 4 µg 4 µg P1 P2
Expression analysis
Final Products
37 50 75 100 150 250 kDa
100 150 250 kDa
E T S M
kDa M T S E
1 µg 2 µg 5 µg 10 µg
100 150 250 kDa
M
15 20 25 37 50 75 100 10 150 250
75 50 37 25 20 15 10
75 50 37 25 20 15 10
Temperature Titer (mg/L) Time (h)
Optimization
Target
Target
Target
15 20 25 10
Truncated
Case Study: MHC Production ɑ 2 ɑ 1 ɑ 2 ɑ 1 ɑ 2 ɑ 1 ɑ 2 ɑ 2 ɑ 1 ɑ 2
ɑ 1
ɑ 1
Over 260 MHC proteins produced from E. coli (0.1 L to 50 L)
+ +
+ +
+ +
Major histocompatibility complex (MHC) proteins are ternary complexes consisting of three components. They are notorisouly hard to express and assemble, while retaining the proper peptide presenting function. MHC Complex HLA B2M Peptide MHC Biotinylation ɑ 3 ɑ 3 MHC Complex HLA B2M Peptide MHC Biotinylation ɑ 3 ɑ 3 MHC Complex HLA B2M Peptide MHC Biotinylation ɑ 3 ɑ 3
Purification
Purification
Purification
ɑ 2 ɑ 2
ɑ 1 ɑ 1
ɑ 2 ɑ 2
ɑ 1 ɑ 1
ɑ 2
ɑ 2
ɑ 1
ɑ 1
ɑ 2
ɑ 2
ɑ 1
ɑ 1
ɑ 2
ɑ 1
ɑ 2
ɑ 1
MHC Biotinylation MHC Biotinylation Biotinylation MHC Biotinylation MHC Biotinylation
Purification Purification Purification Purification Purification
+ + + +
ɑ
ɑ 3 ɑ 3
ɑ 3
ɑ
ɑ 3
ɑ 3
3 ɑ
ɑ 3
3
ɑ
3
3
HLA B2M Peptide HLA B2M Peptide HLA B2M Peptide HLA B2M Peptide
MHC Complex MHC Complex MHC Complex MHC Complex MHC Complex
Purification Profile Purification Profile
Mass Spectrum Mass Spectrum
Final Product
Purification Profile
Purification Profile
Mass Spectrum
Mass Spectrum
Final Product
M 1µg 2µg 5µg 10µg Final Product Final Product
3 x10
3 x10
3 x10
2 x10
2 x10
2 x10
M 1µg 2µg 5µg 10µg
M 1µg 2µg 5µg 10µg
mAU
mAU
mAU
+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr Purification Profile
+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr
Mass Spectrum Mass Spectrum Mass Spectrum Mass Spectrum Mass Spectrum
Purification Profile Purification Profile Purification Profile Purification Profile
3 x10
Q HP Gradient
Q HP Gradient
Q HP Gradient
2 x10 2 x10
mAU 37421.48 mAU mAU mAU mAU
3000 2500 2000 1500 1000 500 0
3000 2500 2000 1500 1000 500 0
3000 2500 2000 1500 1000 500 0
+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr +ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr
+ESI Scan (4.497- +ESI Scan (4.49 +ESI Scan (4.497 +ESI Scan (4.497-5 +ESI Scan (4.497-5
3 x10 1027.4328 3 x10 3 x10
2 x10
2 x10
37421.48
37421.48
1027.4328
1027.4328
0 1 2 3 4 5
0 1 2 3 4 5
0 1 2 3 4 5
8
8
8
2 x10
3 x10 Peptide
Peptide
Peptide
Q HP Gradient
3000 2500 2000 1500 1000 500 0 3000 2500 2000 1500 1000 500 0
Q HP Gradient Q HP Gradient Q HP Gradient 6 Q HP Gradient 6 6
37421.48 37421.48 37421.48 37421.48 37421.48
3000 2500 2000 1500 1000 500 0
3000 2500 2000 1500 1000 500 0
10 10
0 1 2 3 4 5
8
3000 2500 2000 1500 1000 500 0
10 1 1
0 1 2 3 4 5
0 1 2 3 4 5
8
8
0 1 2 3 4 5
8
HLDb Biotinylated
HLDb Biotinylated
HLDb Biotinylated
0 1 2 3 4 5
8
B2M
B2M
B2M
6
1028.4333
1028.4333
1028.4333
4
4
4
6
6
HLDb Biotinylated HLDb Biotinylated HLDb Biotinylated HLDb Biotinylated HLDb Biotinylated
6
6
B2M
B2M B2M 1029.4316
B2M 1029.4316
4
1029.4316
4
4
2
2
2
11861.29
11861.29
11861.29
4
4
2
11861.29 11861.29 11861.29 11861.29 11861.29
2
2
0
0
0
2
mL 700
mL
mL
2
0
0
0
100
100
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200
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700
10000 20000 30000 40000 50000 60000 70000 80000 90000 10000 20000 30000 40000 50000 60000 70000 80000 90000 10000 20000 30000 40000 50000 60000 70000 80000 90000
1026 1027 1028 1029 1030 1031 1032 1026 1027 1028 1029 1030 1031 1032 1026 1027 1028 1029 1030 1031 1032
0
mL mL
0
0
0
mL
mL
100 100
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mL
10000 20000 30000 40000 50000 60000 70000 80000 90000 10000 20000 30000 40000 50000 60000 70000 80000 90000 10000 20000 30000 40000 50000 60000 70000 80000 90000 10000 20000 30000 40000 50000 60000 70000 80000 90000 Counts vs. Mass-to-Charge (m/z) 10000 20000 30000 40000 50000 60000 70000 80000 90000
1026 102 1026 10 1026 102 1026 1027 Co 1026 1027
0
200 Counts vs. Mass-to-Charge (m/z)
100 Counts vs. Mass-to-Charge (m/z)
300 Counts vs. Mass-to-Charge (m/z) 0 300 200 100 0
700 Counts vs. Mass-to-Charge (m/z) 600 600
700 Counts vs. Mass-to-Charge (m/z)
Counts vs. Mass-to-Charge (m/z)
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0
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Counts vs. Mass-to-Charge (m/z) Counts vs. Mass-to-Charge (m/z) Counts vs. Mass-to-Charge (m/z) Counts vs. Mass-to-Charge (m/z)
Co C C
Co
Learn more: Protein Sciences: Early-Stage R&D E. coli Platform
About WuXi Biologics
WuXi Biologics is a leading contract research, development and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.
The world’s leading global single-source platform from concept to commercialization.
wuxibiologics.com | info@wuxibiologics.com
5-1-2024
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