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
100
200
200
200
300
300
300
400
400
400
500
500
500
600
600
600
700
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
200 200
300 300
400 400
500 500
600 600
700 700
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)
400
400
500
500
0
100
200
300
400
500
600
700
0
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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