Case Study: GPCR Production
Purified protein, SDS-PAGE, SEC-HPLC
Aim • Production of a high-quality GPCR protein in insect cells • Reconstitution of GPCR into nanodiscs Key Challenges • Expression level of GPCR was very low <0.2 mg/L • The protein was unstable and its conformation was very flexible • It was hard to reconstitute GPCR into nanodiscs Results • Expression constructs were optimized • ~1.5 mg/L was achieved after optimizing expression and purification protocol • Purity >90% • GPCR’s conformation was stabilized by suitable ligand • Successful assembly of GPCR-nanodiscs
Construct 1: Nt_BRIL
BRIL
ECL1
ECL2
ECL3
N-term
Extracellular
Nanodisc
W P P
Q V
Extracellular
S
Q
A Y N T I L S C O V C N
F
R
D M V Y A A Y C L L L I L I A F L N O V L V A I R E C W M L I P A
C
S I W V A L A Q L L V N L V A
F I F I O L A M M V A T L I N A C V S D
F V V Y M P A T L V
L N P
V L
Y O
P H
L N
Y V
S Y
C P
H I
F A
S Y
S F
S T F S A F V L M V L T Y A Y I V R Q L R I Q R V V
I T F P F A L C C I V F L G V V A M L T Q K K A M E F K K M S
A I F L S W V V M I L L R V R P S A
W Y L
Membrane Scaold Protein
V G L S N A N L
P L
4.5-5.6 nm
V P
Y I T F T N
F
Intracellular
Y N Y
~10.6 nm
T
L
R
A
L
K
M A V
M
T
K
F
A
Intracellular
A Q R A L
F
I
F
ICL2
ICL1
C-term
ICL3
BRIL
Construct 2: ICL3_BRIL
SEC-HPLC
150
M
Monomer
50 kd 75 kd 250 kd
100
GPCR
50
Aggregation
15 kd 20 kd
MSP
0
2
3
4
5
6
Time (min)
Case Study: Automated 2-step Purification of Kinases
Aim • Production of 13 of Kinase variants from Baculovirus pellets Key Challenges • Expression level is different for different variants • Urgent timeline Results • Successfully delivered >95% purity proteins in 1 week
NiNTA elution M T S SEC fraction
Flow-through of sample
mAU
2500
2000 1500 1000 500 0
SEC elution
mL
0
50
100
150
200
250
Automated 2-step purification
Automated 2-step purification results
Run time/sample Max. sample/week Manual operation during steps
4 h 16 No
Learn more: Protein Sciences: Early-Stage R&D Insect 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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