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Strategies to Obtain High-Quality
Recombinant Proteins
- Protocols and Case Studies
Recombinant Protein Expression Overview
Key Influencing Factors for Protein Expression
FAQs & Case Studies
Contents
Recombinant Protein Expression
Overview
1
Applications of Proteins
Protein
Therapeutics
Therapeutic
Antibodies
Structural
Studies
Cell
CultureEnzymes
Immunogen，Function Investigation，Industrial-scale Enzyme ……
Natural Proteins vs. Recombinant Proteins
Natural
Limited resources
Animal contaminants
Low protein yield
Batch-to-batch variations
Recombinant
Recombinant technology
Animal-free
High protein yield
Controlled batch-to-batch variations
Gene synthesisVector
constructionTransfection
Protein expression
Protein purification
QC control
Process for Recombinant Protein Expression
Key Influencing Factors for
Protein Expression
2
How to Obtain Recombinant Proteins
Vector / Host
• Sequence
• Vector
• Host cells
Culture
condition
• Culture medium
• Transfection method
• Culture temperature &
duration
Purification
methods
• Supernatant / intracellular
• Lysis method
• Chromatography purificationKey Factors
Usage of Expression Host
Bill RM. Front Microbiol. 2014
Yeast, 20%
E. coli, 30%
Mammalian cell, 50%
Bill RM. Front Microbiol. 2014
Expression Hosts Used in Bio-
pharmaceutical Area
Expression Systems
E. coli YeastBaculovirus-Insect
cells
Mammalian
cells
• Low cost
• Rapid expression
• Easy to scale-up
• Most widely used
• Rare post-translational
modifications
• Inclusion bodies
• Difficult to express higher MW
proteins
• Low cost
• Rapid expression
• Easy to scale-up
• Some post-translational
modifications
• Unique glycosylation pattern
• High mannose content in
glycan
• Desirable post-translational
modifications
• Easy to scale-up
• High cell density
• Soluble proteins
• More demanding culture
conditions
• Lack of partial glycosylation
• High cost
• Desirable post-translational
modifications
• Soluble proteins
• More demanding culture
conditions
• High cost
Expression Hosts- Post-translational Modifications (PTMs)
Yeast Insect
E. coli Stable CHO
Expression Yield
Yeast E. coli / insect Stable CHO
Low High
Protein Activity
Yeast / Insect Stable CHO / E. coli
Low High
Expression Host Choice- Protein Molecule
Protein type Mammalian cell Insect cell
Secreted protein ++ +
Extracellular region ++ +
Cytoplasm + ++
Viral proteins + ++
Trans-membrane protein + ++
Kinase + ++
Difficult to express ++ ++
Small-MW tags：His, FLAG, HA, Myc
Large-MW tags：IgG-Fc, GST, SUMO
Tag location：N- or C-terminal
Protease digestion sites：EK, 3C, TEV…
Tag removal
Serve as peptide linker
Assisting Protein Expression
Expression Vector- Protein Tag
Facilitate purification
Assist protein folding
Improve solubility
Enhance stability
Increase yield
Prolong protein half-life
Common tags used in Sino Biological
Expression system Tag
HEK293 / CHO His, Fc, flag
Insect His, GST, Fc, flag
Yeast His
E. coli His, GST, Trx, Sumo, MBP, NusA, ZZ
Protease digestion site
Tag Protein Tag
Expression Vector- Protease Digestion Site Selection
Protease digestion site
Enzyme Sequence
Protein Tags Impact Activity
Fc tag His tagFc tag
His tag
Transfection
Vector quality
Transfection reagents:
Liposome, positive ion, etc.
Cell status
Plasmid-to-cell ratio
Culture medium: Ingredient and feed liquid
Culture method:
Stirring: shear force, foam
Dissolved oxygen
pH
Culture temperature & time
Culture Condition
Culture time affects multiple aspects of protein expression
Loss and degradation of plasmid during culture
Protein accumulates along with culture
Track expression status
Protein aggregation and degradation also increase during the course of culture
Culture Time Optimization
Purification Technology
Cell lysesSolid-liquid
separationExtraction Purification
• Dilatometry
• Membrane breakage with
detergent
• High pressure crushing
• Ultrasonic crushing
• High speed centrifugation
• Deep filter
• Microfiltration
• Ultra-filtration
• Affinity purification: Ni-
sepharose
• Cation exchange
• Affinity purification
• Ion exchange
• Hydrophobic chromatography
• Gel filtration
Secreted
proteins
Intracellular proteins
Membrane proteins
Molecule analysis
Sequence & host
Vector
clone
Pilot expression
Process
optimization
Scale-up
production
Protein QC
52%
19%
8%
6%
12%3%
Human Mouse Rat Monkey Viral Others
0
500
1000
1500
2000
2500
3000
3500
4000
HEK293细胞 昆虫细胞 大肠杆菌 酵母 CHO细胞
Pro
tein
nu
mb
er
HEK293 cell Insect cell E. coli cell Yeast CHO cell
Protein Development at Sino Biological
FAQs & Case Studies
3
FAQs
Low production
Degradation
Aggregation
No activity
Recombinant Protein Expression: Case Studies
Causes Solutions
Low expression level
Aggregation and degradation
Toxic protein
Expression vector
Expression host
Culture condition
…… ……
Protein misfolding and
post-translational modifications
Factors Contributing to Low Protein Yield
Culture medium ingredient
293ft cos7
DG44 HepG2
293ft cos7
DG44 HepG2
Sino Biological Leading Brand
3-10-fold higher
Low Protein Yield- Vector Promoter Optimization
Low Protein Yield- Vector Promoter Optimization
Pro
tein
Pro
du
cti
on
(m
g/L
)
Leading
brand 1
Leading
brand 2
Plasmid 1
from Sino
Plasmid 2
from Sino
Plasmid 3
from Sino
MOI adjustment: Improve protein expression
Low MOI: multiple rounds of transfection
High MOI: most cells are transfected once
Adjust culture time
Focus on protein stability
Low Protein Yield- Transfection Ratio
0
10
20
30
40
50
60
70
A B C D E F
sf9 hi-5
Protein expression level in different host cellsCommonly used insect cell lines
Sf9, sf21, high five
Sf9: virus amplification
High five: higher expression level
Low Protein Yield- Expression Host Cell Line
Cell density
Cell viability
Post-modification
Low Protein Yield- Culture Medium Selection
Leading Brand 1
Leading Brand 2
Leading Brand 3
Sino Biological
Pro
tein
Pro
du
cti
on
(m
g/L
)
Culture time post-transfection (days)
Add anti-aggregation reagents to
the culture medium to increase
protein yield
Protein 13-fold yield increase
+- +-
Protein 23-fold yield increase
Low Protein Yield- Reduce Aggregation
Transient transfection
• Short production cycle（within 1 week）
• Sustainable expression within 4-10 days
• Plasmid easily lost
• Batch-to-batch variations
Stable cell line
• Long production cycle
• Plasmids integrate into chromosomes
• High expression level
• Stable protein expression
Secreted protein production: CHO stable cell line > 293 transient transfection
Membrane protein and intracellular protein production: little difference
Transient Transfection vs. Stable Expression
Degradation
Sequence mutation
Expression host
Culture temperature
Culture duration
Purification condition
Protease
inhibitors
Protein Degradation Monitoring and Management
SF9 High five
Mk protein has very low degradation in
High five cell
Degradation Comparison
Kaneda N et al. J Biochem. 1996 Jun;119(6):1150-6
Reduce Protein Degradation- Change Expression Host
High
+++
+++
Low
+
+
Low
++
++
Full length
Fragment
Temperature
Time
Extent of degradation
Reduce expression time
Reduce Protein Degradation- Culture Condition Optimization
Choosing the right cell lysis method
Control shear force, control foaming
Choose the right buffer system
Can only be explored during experiment
Disulfide bond mediated aggregation: add appropriate reducing agents
Optimize purification conditions with aggregate removal
Optimize molecule structure: mutate sites with hydrophobic amino acids
Solutions for Protein Aggregation
Before
After
Culture medium optimization Purification condition optimization
Reducing
reagent - + - +
Non-reducing gel
Solutions for Aggregation Reduction- Culture and Purification Optimization
Culture A B C C
Purification I I I II
Protein tag
Solubility enhancing tag 1
No activity
Solubility enhancing tag 2
Active
Activity is the Key to Protein Applications
Culture and purification
optimization
en
zym
atic
activity
Releasing large amounts of protein and nucleic acids after cell disruption
Protease: degrade target protein
Nucleic acids: long-chain DNA and RNA
Like a glue, it forms a sticky substance that can not be removed via centrifugation, filtration,
and column purification.
Bind to target protein as a form of contaminant.
Hybrid protein: protein aggregation
Target protein form aggregation with itself
Aggregation between the target protein and hybrid protein
Reduce target protein binding to columns
Difficult to remove and cause reduced purity for target protein
Challenges for Intracellular and
Membrane Proteins
Add nuclease after cell disruption
A small amount of nuclease can cleave long-
chain nucleic acids into small fragments
High activity, small quantity of enzyme needed
Low cost
SuperNuclease from Sino Biological
Before After
Solution for Nucleic Acid Contamination
Explore of protein extract
（detergent , pH, salinity, etc.)
After1st
Purification
2nd
purificationTotal proteins Before
Protein extractionSeparate protein from membrane
Solid-liquid separationRemove particles
Crude purificationExtract target protein
PurificationRemove impurities, improve purity
Explore of purification methods
（pH, salinity, buffer and purification column)
Membrane Protein Purification
Key Factors
Expression
host
Sequence
Signal peptide
Protein
expression
Cell culture
Transfection method
Culture temperature
Culture time
Protein
purification
Formulation
screening
QC control
Vector
TagCleavage enzyme Promoter
Key Factors
Expression
host
Sequence
Signal peptide
Protein
expression
Cell culture
Transfection method
Culture temperature
Culture time
Protein
purification
Formulation
screening
QC control
Vector
TagCleavage enzyme Promoter
No One-Size Fits All
12,000+
Antibodies
6,000+
Proteins
500+
ELISA Kits28,000+
Genes
Emerging Leader in Biological
Reagents & Services
Largest Inventory Worldwide,
100% Made in House
• Recombinant Protein Expression Service
One-stop & High quality
CRO Services
• Recombinant AntibodyProduction Service
Sino Biological – Accelerates your Research
5 expression systems Preferred provider for top 10 Pharmas
Contact Us
Headquarter - Beijing, China Branch - PA, US
Tel: +86-400-890-9989
Address: Building 9, Jing Dongbei Technology Park,
No.18 Kechuang 10th St,
BDA, Beijing, 100176, P.R.China
Tel: +1-215-583-7898
Address: 1400 Liberty Ridge Drive, Suite 101,
Wayne, PA 19087
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