E. coli Recombinant Protein Production

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

96DWP, >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

The challenge was to perform an expression optimization study to improve protein expression.

Key Challenge: Low expression

Expression condition optimization

Parameters

Original

Optimized

Expression Analysis

Final Products M 4ug 4ug P1 P2

Expression analysis

Final Products

Medium Strain

Platform Medium BL21 (DE3) 20 1-5 24

Optimized Medium BL21 star (DE3) 37 >100 24

37 50 75 100 150 250 kDa

100 150 250 kDa

E T S M

kDa M T S E

1ug 2ug 5ug10ug

M

100 150 250 kDa

15 20 25 37 50 75 100 10 150 250

75 50 37 25 20 15 10

75 50 37 25 20 15 10

Optimization

Temperature Titer (mg/L) Time (h)

Target

Target

Target

15 20 25 10

Truncated

Case Study: MHC Production ɑ 2 ɑ 1 ɑ 2 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

Purification

ɑ 2

ɑ 1

ɑ 2

ɑ 1

+ +

MHC Biotinylation

Purification

ɑ

ɑ 3

3

HLA B2M Peptide

MHC Complex

Purification Profile

Purification Profile

Mass Spectrum

M 1ug 2ug 5ug10ug Final Product

3 x10

2 x10

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

Purification Profile

Mass Spectrum

Q HP Gradient

3 x10

2 x10

mAU

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 (4.497-5.7

37421.48

1027.4328

0 1 2 3 4 5

8

Peptide

Q HP Gradient

3000 2500 2000 1500 1000 500 0

37421.48

102

0 1 2 3 4 5

8

6

HLDb Biotinylated

B2M

6

1028.4333

HLDb Biotinylated

4

B2M

4

1029.4316

2

11861.29

2

11861.29

0

ml

0

0

100

200

300

400

500

600

700

ml

10000 20000 30000 40000 50000 60000 70000 80000 90000

1026 1027 1028 1029 1030 1031 1032

0

100

200

300

400

500

600

700

10000 20000 30000 40000 50000 60000 70000 80000 90000

1026 1027

Counts vs. Mass-to-Charge (m/z)

Counts vs. Mass-to-Charge (m/z)

Counts vs. Mass-to-Charge (m/z)

Cou

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

2-7-2023

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