Recombinant Protein Service Handbook [2022 edition]

www.genscript.com

Make People and Nature
Healthier Through Biotechnology

ABOUT US
GenScript Biotech Corporation (stock code: HK.1548) is a leading global provider of life
science research, development and manufacturing services. Rooted in solid gene synthesis
technology, GenScript has established four major platforms: life science service and productplatform, biomedical contract development manufacture organization (CDMO) platform, celltherapy platform and industrial synthetic biological products platform.
Founded in 2002, GenScript established its R&D and manufacturing headquarters in Nanjing,
China in 2004. In 2015, GenScript was listed on the Main Board of the Stock Exchange of Hong
Kong, with legal entities in the United States, China, Hong Kong, Japan, Singapore, theNetherlands and Ireland. It operated business in over 100 countries and regions worldwide,
providing quality, convenient and reliable services and products for more than 100,000
customers.
As of December 31, 2021, GenScript owned more than 5,200 employees worldwide, with over
40% of them holding a Ph.D. or master's degree. GenScript has a number of intellectual
property rights, including more than 180 granted patents and more than 670 patent applications,as well as a high dense technical secrets.
With its mission of “making people and nature healthier with biotechnology”, GenScript iscommitted to be one of the most trusted biotechnology companies in the world. As of December
31, 2021, GenScript’s services and products have been cited in over 65,600 peer-reviewed
international academic periodical articles.

HISTORY & MILESTONES

2014
Established Legend Biotech(Cell Therapy Division)Awarded the Commissioned
Research Institute Leadership
Award

2011
2004
Introduced custom protein andantibody services
Established research and production
facilities in Nanjing, China

Established a new R&D and production
base in Nanjing
Participated in the synthetic yeast
genome Sc2.0 project as the only
commercial entity selected
Established Japanese subsidiary
Number of employees reached 1,000

2015
Listed on the Hong
Kong Stock Exchange(stock code: HK.1548)

2002
GenScript established in New
Jersey, USA

2009

2013

Received investment fromKPCB China/The Balloch
Group

Established Bestzyme(Industrial Synthetic
Bioproducts Division)

2019
2017
Legend Biotech and Janssen Biotech enteredinto a global strategic partnership for BCMA
products
CFDA accepted Legend Biotech's IND
application
Acquired 100% shares of CustomArray andobtained chip gene synthesis technology

2018
BCMA Product received IND approval in
China and U.S.
BCMA program progressed well in China
and U.S.
Biologics CDMO business unit officiallyestablished

New GMP Biologics CDMO R&D Center was in operation
LCAR-B38M/JNJ-4528 was granted orphan drug status by the FDA
and priority drug status by the EMA
LCAR-B38M /JNJ-4528 U.S. Phase 1b /2 clinical data achieved
excellent performance of 100%ORR and 69%CR

2020
The Company jointly developed
the world's first neutralization
antibody detection kit with
Singapore to combat the
epidemic of COVID-19
Legend Biotech was publicly
listed on NASDAQ

2021
GenScript ProBio (Biologics
CDMO) became a leading
CDMO in gene therapy and celltherapy in China through A
round of financing
Legend Biotech Cilta-cel celltherapy product submitted abiologics license application(BLA) to the FDA and expected
to be approved by the end of
February 2022
With more than 5200 employees, the group distributed life
science production capacity in
Singapore and the UnitedStates and cell therapy
production capacity in Belgium
to serve the global market

Content

01
02
03
04

Recombinant Protein Expression Platform
Prokaryotic Protein Expression System

04

Baculovirus-Insect Cell Expression System

08

Mammalian Cell Expression System

10

Cell Line Development Platform
Lentivirus Packaging Service

15

CellPower™ Cell Line Development Service

18

Resource Center
Technical Resources

23

Bioinformatics Tools

24

FAQ

25

Literature Published by Customers

27

Order Method and Order Query
Order Method

30

Order Query

30

01

Recombinant Protein
Expression Platform

Recombinant Protein
Expression Platform

Protein Production Capacity
Successful delivery of >40,000 batches of high-quality protein
Successful delivery of >30,000 batches of recombinant antibody
Success rate >98%

Successful delivery of multiple proteins
Transmembrane Protein

Antibody

KinaseNuclear Hormone
Receptor

Co-expression
ProteinEnvelope Protein

Cell Factor

02

Growth Factor

Expression Platform
Recombinant Protein

Application Range
GenScript has over 18 years of experience in protein expression and purification. The Company combines
an experienced production team with advanced equipment to ensure that it can meet the different needsfrom a variety of customers, including complex projects such as protein expression in multiple systems
followed by purification and specific analysis.
Target Identification andVerification
Enzyme Identification
High Throughput Screening

Antibody Preparation
Structural Biology
Research
Eukaryotic Protein Analysis

Isotope Labeled Protein Tag
Research
SAR (Structure-Acitivity Relationships)

Workflow

Verisign
SecuredSubmit gene sequence by email

Gene synthesis and subcloning

Delivery of expressed
protein samples

Protein expression andpurification

QC by multiple methods

03

Recombinant Protein Expression Platform

Prokaryotic Protein Expression System
The prokaryotic protein expression system is both commonly used and economical. Represented by theE. coli expression system, it has the advantages of clear genetic background, low cost, high expression
and relatively simple separation and purification of expressed products.

Service Type
BacPower™ customized protein expression service
Bacterial fermentation service

Service Process
Preparation of competent cells

Strains cryopreservation

Plasmid preparation

DNA

Competent cell

Transformation

Cell culture

Purification and detection

Prokaryotic Protein Expression System

04

Customized services are available for proteins with special experimental needs such as structural analysis
and functional analysis, and customers can provide experimental protocols and intermediate samples.
GenScript offers customers with flexible services to meet different needs: from codon optimization to genesynthesis, to small-scale testing and amplification, as well as protein purification and property analysis.

Service Advantages

Flexible

Fast cycle

Multiple customization options

Service Content
Milestones

Specifications

1

Gene synthesis and Codon optimization

Gene synthesis, Cloning,
Plasmid Prep

Subcloning into appropriate expression vector

2

Transform plasmids into appropriate bacterial
expression strain

Protein Expression
Evaluation

3

4
QC & Delivery

Timeline*≤2 weeks

Plasmid Prep and DNA QC≤1 week

Protein expression evaluation and optimization

Scale up protein expression using optimized
conditions
Protein Purification, Tag
Removal (Optional) &Refolding (Optional)

Deliverables

Purification to reach desired protein amounts
and purity
Tag removal and separation of tag-free protein(if requested)

Optimized gene sequencereportProtein in buffered solution
with your specified amount &purityQC data≤1 week

Refolding in case of insoluble protein(performed only if required)

Purity analysis and Detection using SDS-PAGE
and Western Blot (for tagged protein)
Bradford assay for quantitation≤1 week

Protein delivery

Note:*All timelines are approximate. In most cases, we deliver within 4-6 weeks. However, optional steps might prolong the overall timeline slightly.
For any large scale protein production requirements, you could try our fully customizable E. coli fermentation services. Talk to our technical account managers for more details.

05

Recombinant Protein Expression Platform

BacPower™ customized protein expression service

Case 1: Protein expression
SDS-PAGE & Western Blot Analysis
Western BlotSDS-PAGE
kDa

M1 BSA R

M2

250

R

120

150
100
75

80

50

50

60

42

37

M1: Protein Marker
M2: Protein Marker
BSA:2 μg
R: Reducing condition

32

25
20
15
10

18

Case 2: Professional refolding service

30
20
10

Human BMP9 12.2 kDa
BSA and target protein: 2 μg

M

R

5

10

kDa
120

10

Human MHC I HLA 34.7 kDa

158 kDa, 9, min

670 kDa, 7.1, min

20

mAU

80
60

30

Time (min)06

17 kDa, 15.1, min

1.4 kDa, 17.7, min

20

VWD1A, Wavelength=280 nm VWD1A, Wavelength=280 nm

NR

40

15

1.4 kDa, 15.8, min

40

44 kDa, 13.6, min14.4, min80
60

13.2, min

kDa
120

100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0

158 kDa, 11.4, min

VWD1A, Wavelength=280 nm VWD1A, Wavelength=280 nm

NR11.2, minR

17 kDa, 12, min

BSA

670 kDa, 9.1, min

M%

Recombinant Protein Expression Platform

Cases

25

High purity protein is a prerequisite for scientific research such as high-throughput screening, functional
analysis, and structural biology. However, large-scale protein production and purification is not only abottleneck for some scientists, but also requires some core equipments.
GenScript provides industrial scale bacterial fermentation services to meet customers’ needs for specificprojects, with fermentation capacity up to 2,000 L.

Service Advantages

Flexible fermentation
capacity of 10-2,000 L

Multiple large-scaleprotein purification methods

Gene-to-protein
integrated services

Cost-effective

Service Content
GenScript bacterial fermentation services are tailored to the needs of customers, from optimization ofprotein expression, microbial culture conditions, and scale-up cultures, to large-scale production, protein
purification, and identification. The fermentation capacity is up to 2000 L. GenScript can provide you withup to 3 tons of transformed bacterial cytoplasm (cell pellet) or gram level recombinant proteins with purity
up to 98%.

Delivery of Products
Bacterial cytoplasm (cell pellet)
Batch record
QC report

07

Recombinant Protein Expression Platform

Bacterial fermentation service

Recombinant Protein Expression Platform

Baculovirus-insect Cell Expression System
The baculovirus-insect cell expression system is a powerful and commonly used system, which is suitable
for the preparation of large-scale recombinant proteins. It is considered as a potent tool for protein expression because of its simple process, high expression level, and post-translation modifications.
Covering virus preparation and large-scale protein production, GenScript baculovirus protein expression
system provides corresponding services for pharmaceutical companies, biotechnology companies, andacademic research institutions. Based on the codon preference of Sf9, Sf21, Hi5 and other insect cell lines,
GenScript provides free codon optimization for you to effectively improve protein expression.

Service Advantages

Success rate forprotein expression
above 90%

Multiple expressing cell
(Sf9, Sf21, Hi5 and others)

Fast delivery ofplasmid to protein

One-stop service fromgene to protein

Service Workflow
Promoter
Tn7R

GOI

Tn7L

Transformation
Target gene

HelperDonor
plasmid

BacmidDH10Bac competent cell

HelperTransposition
Antibiotics
screening

E.coli containing recombinant bacmids

Bacmid extraction
Recombinant virus particle

Determination of virus titration by qPCR

Bacmid transfection

Protein expression confirmationand virus extended culture

Insect cell
infection

Virus stock

Baculovirus-insect Cell Expression System

08

Recombinant baculovirus DNA

Delivery

Cycle

Construct(s) report

2 weeks

Specifications

1

Subcloning into one or more expression vectors

Gene synthesis andsubcloning

Recombinant Protein Expression Platform

Milestones

Generation of recombinant Bacmid DNA
Transfection of insect cell with recombinant Bacmid DNA

2

5 ml, >107 pfu/ml

Generation of P1 stock (Low titer), P2 stock (High titer),and determination of virus titer by Quantitative-PCR

Virus generation

2~3 weeksvirus stock

Verify the protein expression for P1 and P2 generation bywestern blot with anti-His/GST or other antibody

3
Pilot expression
evaluation

4
1 L expression
and purification

Infection of insect cells with P2 stock
One step affinity purification
SDS-PAGE gel and Western blot

Expression data

1.5 weeks

1 L insect cell expression and purification
One-step purification
QC by SDS-PAGE, Western blot2, and LC-MS/MS coverage2

Purified proteins

1~2 weeks

QC dataNote:
1. Listed is the typical turnaround time at GenScript. Please note that additional requirements may incur additional steps, and likewise longer processing time.
2. Other QC data, such as LC-MS/MS coverage, N-terminal sequencing and LC-MASS are available with additional charge.

Cases
Case 1: Kinase protein expression
kDa

A Virus

M

120
80
60

Kinase

40
30

20

20
10

Sf9 cells expressed kinase with 95% purity and yield to 10 mg/L.

Case 3: Macromolecule weight protein expression

120
80
60

kDa
120
80
60

40
30

10

kDa

Case 2: Virus protein expression

Non-reducing
M

Reducing

kDa
200

g

cin

B Virus

du
g
Re
cin
du NonRe

116
97
66
44
29
20

The virus protein was successfully expressed in
the insect baculovirus system.

Case 4: Multisubunit complex protein
5-subunit complex

4-subunit complex

3-subunit complex

Dimer, 200 KDa
Monomer, 100 KDa

40
30
20
10

Hi5 cells expressed macromolecule weight protein(100 kDa) with 95% purity and yield to 10 mg/L.

The multisubunit complex protein was successfully
expressed in the insect baculovirus system.

09

The mammalian cell expression system has the process of both post-translational modifications and functional folding during the protein expression. This feature allows the expressed protein closer to the naturalstate of mammalian proteins in terms of molecular structure, physical and chemical properties and biological function, with the necessary spatial structure and modification, and resulting in the specific biologicalactivity. It also makes the mammalian cell expression system more widely used in the development andproduction of functional proteins, antibodies and clinical vaccines.

Service Type
TurboCHOTM high throughput expression (TurboCHOTM HT)
Expression of gram level protein (TurboCHOTM Express, TurboCHOTM HP)

Expression service of high-throughput antibody (CHO-HT)High throughput expression is a cost-effective option for producing small-scale recombinant antibodies andproteins. GenScript HTP service is a one-stop service from gene synthesis to purified antibody/protein, and
we look forward to assisting your research at full speed!
From gene synthesis to protein or antibody, fast delivery takes only 2-week, and triple QC testing canensure the delivery quality! The whole process is highly automated, the samples are monitored by therespective QR codes, and the whole process can be traced. Coordinating with the Biosecurity Law, GenScript can provide you with comprehensive support including materials in a more standardized and comprehensive manner for your downstream R&D projects.

Service Advantages

Fast delivery in
2-weekTraceability of thewhole process

Comprehensive
improvement of the delivery

Multi-step QC

Service Process
NH2

Tyr
Gly
Gly

Phe

Met

Arg

e
tanc
sis

MCS

AB R
e

Recombinant Protein Expression Platform

Mammalian Cell Expression System

Arg
Val
CONH2

Antibody sequences
provided bycustomers

10

O riginCodon optimizationHighfor sequence
throughput
analysisgene synthesis

High throughput
antibody
expression

Purification
and QC testing

Delivery yourrecombinant
proteins

Recombinant Protein Expression Platform

Service Content
Volume

1 mL

4 mL

20 mL

Timeline

Fast delivery in 2-3 week

Cell line

CHO

Minimum order
quantity

8

Protein types

Standard antibody, antibody fragment (customized recombinant protein), bispecific antibody

QC*

SDS-PAGE, endotoxin control and sampling inspection (other customized QC items)

Process

Gene synthesis to antibodies (customized recombinant protein)

Purification

One step purification (customized multi-step purification)

Additional services

Multiple ELISA, mass spectrometry, affinity determination, phenotypic mapping, cross reaction, in vivo/in vitro experiments

QC*: LC-MS, ELISA analysis, quantitative detection of endotoxin, peptide mapping and other QC methods;
Antibody type: chimeric antibody/Fab/scFv/bispecific antibody/class switching antibody/nanobody, etc;
Downstream expansion experiment: affinity experiment, cross-reaction experiment, in vitro and in vivo efficacy, epitope analysis, stability testing, sterility testing, etc.

Expression of gram level protein (TurboCHO Express, TurboCHO HP)
TM

TM

GenScript developed a new generation of proprietary CHO transient expression that significantly increased
yields and reduced production time. CHO transient expression is a cost-effective protein expression
scheme that enables small-scale (microgram) to kilogram-scale protein production with good consistency indownstream applications.

Service Advantages

High expression titer

Validated consistency

Easy to scale up

Service Process
High efficiency
transfection

Expression

Proteinproduction

11

Recombinant Protein Expression Platform

Service Content
CHO-Express

CHO-HP

Demanded quantity of protein

mg～g

g～kg

Expression volume

40 mL～200 mL

200 mL～200 L

Timeline

3 weeks

6 weeks

Deliverables• Purity: ≥ 95% (antibody)

Default QC testing
Additional QC testing• Endotoxin: ≤ 1 EU/mg (antibody)

SDS-PAGE, SEC-HPLC, endotoxin, A280, LC-MS (only for antibody)Affinity experiment, Cross reactivity experiment, epitope classification, in vitro and in vivo efficacy,
epitope analysis, sterility testing, etc.

Purification

Multi-step purification available

Cases
Case 1: Fc fusion protein expression
VHH
or recombinant protein

CH2

CH2

Fc region

SDS-PAGE

Lane M1: Protein Marker
R: Reducing condition
NR: Non-reducing condition

SEC-HPLC

CH3

CH3

Molecule 1: VHH + Fc

Molecule 2: Recombinant protein + Fc
SDS-PAGE

Lane M1: Protein Marker
R: Reducing condition
NR: Non-reducing condition

Product purity > 99% by SEC-HPLC

12

SEC-HPLC

Recombinant Protein Expression Platform

Case 2: Expression of asymmetric bispecific antibody
SDS-PAGE

M

R

SEC-HPLC

NR

VH

VL

VH

VL

200 -

CH1

CL

CH1

CL

116 97 66 -

CH2

CH2

44 -

CH3

CH3

20 14 -

29 -CrossMab (KiH backbone)

The expression of assymmetric bispecific antibody was
299 mg/L, and the purity was 99% by SEC-HPLC

Contact US and send email to protein@genscript.com

13

02

Cell Line Development PlatformLentivirus, a type of retrovirus, has become an essential tool for gene transduction. Lentiviruses can introduce target genes into cells that are difficult to transfect, such as primary cells and stem cells, thus greatly
improving the transfection efficiency of target genes. Lentiviruses are also widely used in the constructionof stable cell lines.
GenScript introduces lentivirus packaging services with diverse titers and specifications, flexible options
and fast delivery (as fast as 2 weeks).

Service Advantages

Cost-effective

Fast delivery

Strict quality control

Less cost for the same amount of virus

As fast as 2 weeks

Stringent testing standards

Service Process

Cell culture

Transfection

Virus collection

Purification

Titer testing

Service Content
Service

Titer

Specification>107 IFU/mL

1 mL>107 IFU/mL

2 mL>10 IFU/mL

1 mL

p24 ELISA>108 IFU/mL

2 mL

Mycoplasma test

>109 IFU/mL

0.1 mL>109 IFU/mL

0.2 mL

8

Lentivirus packaging(SC1394-VP)

QC standard

Cycle

2~3 weeks

Customized services
For customized services, please send an email to protein@genscript.com

15

Cell Line Development Platform

Lentivirus Packaging Service

Case 1: Lentivirus transductional performance
GenScript

Competitor 1

Competitor 2

EGFP lentivirus was packaged by GenScript, Competitor 1 and competitor 2, respectively. After the determination of titers, they were
adjusted to the same MOI to infect the same amount of 293T cells. Fluorescent photographs were taken 72 hours after infection.
GenScript lentivirus has the highest transductional efficiency.

Case 2: GFP lentivirus infection Jurkat MOI testing
MOI=20

count

count

MOI=10

MOI=40

MOI=80

count

count

Cell Line Development Platform

Cases

MOI

GFP

Infection time

10

17.1%

72 h

20

26.1%

72 h

40

41.6%

72 h

80

60.1%

72 h

Jurkat cell is a type of T lymphocyte that is difficult to transfect. Jurkat cells were infected with GenScript third-generation GFP lentivirus packaging plasmid system and tested to achieve an infection rate of 60.1% at MOI=80.

16

Cell Line Development Platform

Case 3: Optimized vector performance
Infection efficiency
Commercial Vector

Cell line

GS Optimized Vector

120.00%

Name

MOI

CT26

Mouse colony

BAF3

Mouse B cell

EL4

Mouse T lymphoma

RAW 264.7

Monocyte/macrophage-like cells

JurkatHuman acute T cell leukemia

20.00%

K562

myelogenous leukemia cell line

0.00%

NALM6

B cell precursor leukemia cell

THP-1

Acute monocytic leukemia**
100.00%80.00%******
60.00%
40.00%**

100

MOI: Multiplicity of infection

Our optimized vector can help you to achieve higher infection rate even in hard-to-transduce cell line.

Case 4: GenScript lentivirus packaging system with different target gene length testing
Gene name

Target gene length

Titer (×108 IFU/mL)

Gene 1

3.8 kb

3.43

4.7 kb

3.58

Gene 3

5.5 kb

1.84

Gene 46.6 kb

2.61

Gene 2.

In general, when the length of the target gene exceeds 3000 bps, it is difficult to reach a high titer level, and even can not be packaged.
GenScript's lentivirus packaging system can reach up to 6.6 kb. GenScript can guarantee a titer of 108 IFU/mL for conventional genes(moderate GC content, no complex repeats, no cytotoxic gene and less than 5 kb).

Downstream Application
Stable cell line construction, such as overexpression cell line, functional assay cell line and knockdown
cell line.
Construction of gRNA virus library.
Improve the efficiency of gene transduction in diffficult-to-transfect cell lines, such as primary cells, iPS
cells, other stem cells and non-dividing cells.
Provide high-quality lentivirus for animal model experiments.
Clinical treatment research, such as gene therapy, CAR-T cell construction.
Vaccine research.

17

Cell Line Development Platform

CellPowerTM cell line development service
Stable cell lines are widely used in drug discovery, toxicity testing and basic research. Long-term stable
expression of a gene of interest (GOI) is usually achieved by plasmid transfection or virus transduction whichcontaining the expression cassette of GOI together with a selection marker (antibiotics or fluorescentproteins). Recombinant stable cell line development at GenScript includes constitutive expression andinducible expression. In addition, we developed lentivirus based CRISPR/Cas (Lenti-CRISPR/Cas9)
technologies and platforms, in order to meet the increasing need of GOI-knockout cell lines, especially for
difficult-to-transfect cells.

Service Advantages

Multiple transfection
or infection modes

250+ target
cells experience

Extensive protein
expression
experience

180 species of
mycoplasma testingMultiple
detection methods

Service Process

Multiple methods for transfection/
infection optimization
GFP positive control
Gene synthesis, plasmid
construction and virus
packaging

Gene transfection efficiency

Transfection of host

optimization

cells

Sequencing
Transfection grade plasmid
High-titer lentivirus

18

Multiple antibiotics like Puro, Blasticidin
Zeocin and Hygromycin for screening
FACS sorting
Serial or limited dilution to generatemonoclonal cell clones

Screening of cell poolor monoclonal
antibodies

GFP positive control to ensure
successful infection or transfection

Cell bank
Cell viability testing
Mycoplasma testing
COA report

Validation of cell pool

Delivery ofor cell clones

stable cell lines

QC: WB, FACS, qPCR,
sequencing, etc.

Cell Line Development Platform

Service Content
Service number

Delivery

Cycle

Report of cell line testing

CellPowerTM
Constitutive overexpression

2 cryovials of pooled cells with 106 cells/vial

Stable pool: Starting from 8 weeks

Report of cell clone generation

Single clone: Starting from

Report of cell clone screening

12 weeks

2 cryovials of single clone with 106 cells/vial

cell line

COA report

Report of cell line testing
2 cryovials of pooled cells with 1×106 cells/vial
Report of cell clone generation
CellPower

TM

Reporter cell line

Stable pool: Starting from 10 weeks

Report of cell clone screening

Single clone: Starting from

1 cryovials of single clone with 106 cells/vial

14 weeks

COA report/analytical method

Report of cell line testing
2 cryovials of pooled cells with 1×106 cells/vial
CellPowerTM Inducible

Report of cell clone generation

overexpression cell line

Report of cell clone screening

Stable pool: Starting from 10 weeks
Single clone: Starting from

1 cryovials of single clone with 10 cells/vial
6

15 weeks

COA report

In addition to the above lentivirus cell line development services, GenScript also offers CRISPR/Cas9 cellline development services.
CRISPR is a powerful tool for research and discovery of drugs that change multiple genes in cells inhighly targeted ways. Compared with other gene editing forms, such as TALENs and zinc finger nuclease(ZFN), CRISPR/Cas9 has the advantage of being easier to manipulate and more efficient in performing
biallelic modifications.
GenScript provides GenCRISPR™ based gene editing services that allow the development of gene-edited cells for multiple genes using multiple mammalian cell lines.
For more information on CRISPR-related services, please refer to the GenScript Gene-editing
Services Manual.

19

Case 1: Stability testing of reporter gene overexpression

Expression stability test in 30 generation

Reporter gene testing
Stablit y
8 .0 × 1 0

Stablit y

GS-J2 /GOI

4

GOI G815

GS-J2 /GOIGOI G8GOI G13

GOI G13

GOI G18

GOI G18

GOI G23

4

GOI G28
GOI G33

4 .0 × 1 0 4

GOI G23

10

Signal/ Blank

6 .0 × 1 0

Luminesecen ce ( RLU)

Cell Line Development Platform

Cases

GOI G28
GOI G33
5

2 .0 × 1 0 4

00
1 0 -4

1 0 -2

100

Conc. ( μg/mL)

102

1 0 -4

1 0 -2

Conc. (μg/mL)

Reporter gene expression stability test in 33 generation

20

100

102

Cell Line Development Platform

Case 2: Tet on inducible assay cell line

Verification of Gene A mRNA expression by qPCR
Column 1. Hep-G2 parent cell
Column 2. Hep-G2/Tet on/Gene A Dox 0 ng/mL
Column 3. Hep-G2/Tet on/Gene A Dox 500 ng/mL
Column 4. Hep-G2/Tet on/Gene A Dox 1000 ng/mL
Column 5. Hep-G2/Tet on/Gene A Dox 1500 ng/mL
Column 6. Hep-G2/Tet on/Gene A Dox 2000 ng/mL

Verification of Gene A protein expression by Western Blot
Column 1. Hep-G2 parent cell
Column 2. Hep-G2/Tet on/Gene A Dox 0 ng/mL
Column 3. Hep-G2/Tet on/Gene A Dox 1000 ng/mL

Case 3: Tet off inducible assay cell line

Verification of Gene A mRNA expression by qPCR and FACS
Column 1：Colo 205/Tet off
Column 2：Colo 205/Tet off_Gene A 0 ng/mL Doxycycline
Column 3：Colo 205/Tet off_Gene A 100 ng/mL Doxycycline

Downstream Application
Drug screening.

FACS screening of membrane protein antibody.

Gene expression regulation.

Localization studies or live imaging with fluores-

Functional assay.

cence labeled proteins.
In vivo tumor immunity research.

21

03

Resource CenterTechnical Resources
White Paper - 6 Steps to Optimize Your Recombinant
Antibody Expression
This white paper provides technical resources for scientists in the fields ofantibody engineering, therapeutics and research and development, and for
optimizing expression systems.
You will learn from this white paper:
Choosing an expression host system
Designing an expression vector
Optimizing the cell expression system

www.genscript.com

4 Strategies for Boosting Transient
Protein Expression

Choosing between transient expression and stable
expression
Choosing a suitable transfection method
Choosing a suitable purification method

4 Strategies for Boosting Transient Protein Expression
This application note is a free educational and technical resource for biomedical
scientists studying proteins or antibodies.

White Paper

You will learn from this handbook:
Designing an expression vector

Increasing protein stability

Utilizing a fusion partner

Improving protein purification

Recombinant Antibody Handbook - Processes andStrategies
This handbook is designed to help you improve the purity and yield of recombinant antibodies.
You will learn from this handbook:
Recombinant expression and purification of IgM and a bispecific antibody
Help you optimize recombinant antibody expression; transfection & growth conditions;recombinant antibody purification & characterization.
Bioinformatics resources available to aid in the design of recombinant antibody experiments

23

Resource Center

Bioinformatics Tools

FoldArtTM

A patented in vitro soluble and folding platform to recover purified proteins from
inclusion bodies produced by high protein expression, including high hydrostaticpressure, small molecule additives and column folding technology to recoverhigh-purity soluble proteins.

GenSmartTM

GenSmart™ Codon Optimization technology is more accessible and user-friendly.
All key factors have been integrated into the algorithm, which can be completed byfilling in the basic information (such as sequence and host), and customized optimization of each gene leads to a higher probability of obtaining functional and active
proteins.

Solubility Tags

The E.coli expression vector with a unique solubility marker developed by
GenScript's professional scientists is specially used for the production of soluble
recombinant proteins.
The success rate of the E. coli project is greater than 95%.

Contact US and send email to protein@genscript.com

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Resource Center

FAQ
Questions related to protein expression
Q: What are the capacities of GenScript’s protein expression platforms?
A: For Bacteria System: (a) Provides large quantities of protein, up to grams; (b) Provides large-scale fermentations up to 500 L;(c) Provides homogeneous recombinant proteins, over 98% purity.
For Baculovirus-Insect Cell System: (a) Provides high titer recombinant baculovirus stock carrying the genes of interest, up to 108
pfu/mL; (b) Provides homogeneous recombinant proteins, over 98% purity; (c) Provides large-scale protein production, up to
hundreds of milligrams (or 100 liter culture).
For Mammalian Cell System: (a) Provides homogeneous recombinant proteins, over 98% purity; (b) Provides protein production,from miligram to kilogram level (or 200 liter culture).
Q: If the expression level of transient expression is low, how do you usually adjust or optimize theexperimental protocol to improve the expression level?
A: Generally, the protein expression is mainly determined by the nature of the protein itself. The common optimized measurements are as
follows: codon optimization, transfection mode optimization (especially for some cell lines with low transfection efficiency), cell culture
scheme optimization, cell lysis buffer testing for protein secretion and expression analysis, host cell line change, signal peptide change,
expression vector change, cloning strategy (such as Fc labeling strategy and site mutation), buffer screening of purification steps(precipitation, solubility) and addition of protease inhibitors (if protein degradation occurs).

Q: Why will customers choose GenScript for custom protein expression?
A: GenScript provides a wide range of protein services with the following features: (a) Your custom requests are handled byexperienced scientists – we have successfully delivered over 40,000 batches of custom proteins and over 30,000 batches of custom
antibodies; (b) High success rate – our E. coli protein expression system has a 98% success rate; Our other expression systems
also have success rates above the industry average. (c) High capacity – up to 2,000 L bacterial protein production, 100 L insect and200 L mammalian protein production. (d) GenScript has developed mature platforms with advanced technologies (e.g.
OptimumGene™, BacPower™, BacuVance™, and CrystalPro™) to solve your protein puzzles.
Q: Does codon optimization really matter? Is there any difference between codon optimization ingene and protein departments? Does codon optimization promote the solubility of the expressed
proteins?
A: Codon optimization can greatly increase the level of protein expression. GenScript OptimumGene™ is the most cited codon
optimization technology. Thousands of cases have demonstrated the effectiveness of GenScript's codon optimization.
Currently, the same system is used for codon optimization in both gene and protein departments, and codon optimization only
promotes higher protein expression, not solubility.
Q: How much baculovirus would GenScript need to produce the protein if the customer supplied it?
A: If the customers provide baculovirus, they should also provide GenScript with the baculovirus titer and generation of viral stock,
MOI, cell density and cell volume for cell infection. We'll use the relevant formula to calculate how much viral stock needs to be
added to obtain the inoculums required (mL). MOI is defined as the number of virus particles per cell. Usually the P1 and P2 viral
stock is preferred, and the titer of viral stock should be more than 107 pfu/mL. If the amount of virus provided is insufficient, we alsooffer virus amplification service.

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Questions related to protein purification
Q: What are the impurities in protein expression? How does GenScript remove these impurities?
What are the protein purification methods in GenScript? How does GenScript choose a purification
method to use? What is the common process of multi-step purification?
A: Impurities are mostly intracellular or culture medium substances that can be removed by chromatography. GenScript can performaffinity purification, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography Generally, affinity
chromatography is the first choice for proteins containing affinity labels, and then multi-step purification is selected according to theobtained protein purity and the location and properties of impurities .
Q: What does GenScript do during purification if the customer's protein is easily degraded?
A: During the purification process, we will add protease inhibitors and use mild purification methods, such as using cell lysis
enzymes instead of ultrasonication, and will operate quickly at low temperatures to purify the protein in a shorter time. If
necessary, we will use multi-step purification to obtain higher purity of the target protein.

Q: How does GenScript determine protein purity and concentration?
A: Methods for purity determination: (a) SDS-PAGE (b) SEC-HPLC; SDS-PAGE is used as default.
Methods for protein concentration determination: (a) Branford protein assay; (b) BCA protein assay; Bradford is used as default,
and BCA is used if there are any disturbing factors in the buffer. Antibodies are tested with A280.
Q: How are endotoxins controlled? What endotoxin level can GenScript achieve?
A: The prokaryotic system of protein removes endotoxins at three levels: < 1 EU/μg, < 0.1 EU/μg, <0.01 EU/μg. Of which,0.001 EU/μg is difficult to achieve. The 0.001 EU/g for mammalian expression system is easier to reach, and the antibody canreach 0.0005 EU/μg. The typical endotoxic protein requirement for mice is 0.003 EU/μg, and < 0.001 EU/μg for human. The
standard for internal control of mammalian expression is 0.01 EU/mg, but GenScript does not test for endotoxin content bydefault. If the customer requires final QC data including endotoxin, and if the customer has requirements for endotoxin control,
it should be proposed before the experiment. GenScript controls endotoxin levels by managing reagents, containers, to avoid
the introduction of endotoxin during expression and purification. If endotoxin levels are still high, further removal can be donewith a detoxifying column or ion column.

To help you solve problems related to recombinant protein expression and cell line development,
Please visit the FAQs in Technical Resource Center:
https://helpcenter.genscript.com/hc/en-us?src=pullmenu, or contact GenScript Professional Support.

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Resource Center

Literature Published by Customers
Title: Structure of the human cGAS–DNA complex reveals enhanced control of immune surveillance
Journal: Cell IF: 36.216 (2018)
Doi: 10.1016/j.cell.2018.06.026
Introduction: hcGAS (D157–F522) and mcGAS (P147–L507) were codon optimized for bacterial expression.(GenScript)

Title: Ticks Resist Skin Commensals with Immune Factor of Bacterial Origin
Journal: Cell IF: 36.216 (2020-12)
Doi: 10.1016/j.cell.2020.10.042
Introduction: 24 h before tick feeding, 0.5 mg affinity purified Rabbit aDae2 custom generated against recombinant Dae2Is protein(GenScript) was diluted in sterile PBS and injected into the intraperitoneal cavity of female C3H/HeJ mice to neutralize against
pre-made Dae2Is protein in tick saliva.

Title: Human IFIT3 modulates IFIT1 RNA binding specificity and protein stability.
Journal: Immunity IF: 22.845 (2018)
Doi: 10.1016/j.immuni.2018.01.014
Introduction: The coding regions of IFIT1, IFIT2, IFIT3, IFIT5 , ifit1, and ifit3 were codon optimized for expression in E. coli.
(GenScript)

Title: Connecting clusters of COVID-19: an epidemiological and serological investigation
Journal: Lancet Infect Dis IF: 19.966 (2020-04)
Doi: 10.1016/S1473-3099(20)30273-5
Introduction: For ELISA assays, we used recombinant nucleocapsid protein from SARS-CoV and SARS-CoV-2 expressed in
mammalian cell culture using the pcDNA3.1 vector , according to previously published methods and a recombinant receptor binding
domain (RBD) of the SARS-CoV-2 spike protein custom-produced by a commercial provider. (GenScript）

Title: Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin.
Journal: Nat Neurosci IF: 17.839 (2019)
Doi: 10.1038/s41593-019-0366-7
Introduction: Recombinant proteins were generated and obtained from GenScript.

Title: CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.
Journal: Cancer Cell IF: 27.407 (2020)
Doi: 10.1016/j.ccell.2019.11.003 Dec 2019
Introduction: Subsequently, DMSO-conditioned medium or YKL-5-124-conditioned medium were collected and added to above
single-cell suspension in a 96-well U-bottom plate in the presence of Ova257–264 peptide (10 mg/mL, GenScript, Cat#RP10611) for
4 days as previously described.

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Literature Published by Customers
Title: Dendritic cells dictate responses to PD-L1 blockade cancer immunotherapy
Journal: Sci Transl Med IF: 16.761 (2020)
Doi: 10.1126/scitranslmed.aav7431 11 Mar 2020
Introduction: After isolation, DCs were either incubated with DQ-OVA (Thermo Fisher Scientific) or pulsed with OVA peptide 257264
SIINFEKL (GenScript).

Title: An unbiased approach to defining bona fide cancer neoepitopes that elicit immune-mediated cancer rejection
Journal: J Clin Invest

IF: 12.784 (2020)

Doi: 10.1172/JCI142823 1 Feb 2021
Introduction: Peptide synthesis Peptides were custom made with a purity of >90% (JPT, Berlin, Germany and GenScript, Piscataway,
NJ) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM. Generation of BMDCs and neoepitope vaccine
preparation.

In terms of services and products, GenScript have been cited nearly 10,000 times by more than 1300
journals of biomedicine such as Cell, Nature, Science and PNAS.
Please visit the customer's published literature athttps://www.genscript.com.cn/reference_peer-reviewed_literature.html.

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Molecular Biology Services
Catalog Products
Peptide Services
Protein Services
Antibody Services
Oligo Services
Bioinformatics Tools
2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021

Number of Literatures cited GenScriptfrom 2010 to 2021

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Gene Editing Services
COVID19

Proportion Distribution of GenScript
Products and Services in the Literature

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Order Method and Order QueryOrder Method and Order Query

Order Method
Email US: You can send your demand information to protein@genscript.com
Call US: USA: +1-732-885-9188
Netherlands: +31 (0) 71 569 0120
United Kingdom: +44 (1865) 679988
Singapore: +65 3159 1898
Japan: +81-3-6811-6572
Korea: +82-10-9311-9208

Order Query
How to query?

Log on GenScript website

1. Log in to your GenScript account
2. Click Account Name - User Center

User Center

3. Click "My Order/Query" in the taskbar on the left of the page
4. Select "All Types of Orders” in the Order TypeMy Order/Query

5. Click the order number to enter the "Order Details" page to view theorder progress. For delayed or difficult orders, please email us for

Select Service

consultation and confirmation. We will reply and follow up as soon as
possible.

Click the Order Number

For delayed or difficult orders, please email us for consultation and confirmation. We will reply and follow
up as soon as possible.

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