1
Protein expression & purification
technologies
Manufacturing of recombinant proteins, protein complexes
and virus-based vehicles for biotechnology and basic research
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
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Molekulární biotechnologie cvičení
Středa 9. říjen 2019 10:00 – 11:50: A13/332
Středa 9. říjen 2019 14:00 – 15:50: A36/308
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What will we talk about
• Introduction to recombinant protein production
• definition, goals and applications
• Protein expression in microbial cells
• Escherichia coli, yeasts
• Expression and multi-expression in eukaryotic cells
• Mammalian and insect cells, baculovirus expression
• New emerging expression tools
• CRISPR/Cas9 technology, cell-free protein expression
• Protein purification, characterization and storage
• Chromatography separation & QC check
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Why to purify a protein?
• To study its function
• To analyze its physical properties
• To determine its sequence
• To determine its three-dimensional structure
• For industrial or therapeutic applications
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Applications of protein technologies
Why should we bother to think about protein technologies?
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The key role of protein production in pharma industry
Protein production is
a significant bottleneck in early
phase drug discovery
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Recombinant protein production workflow
• Molecular cloning
• Protein expression
• Protein purification
• Protein characterization
• (Protein structure determination)
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Cellular protein production: selection of a host
• Escherichia coli
• Yeasts (Pichia, Kluyveromyces)
• Insect cells (Baculovirus expression system)
• Mammalian cells (HEK293)
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Bacterial expression system
Concepts
Methods
Applications
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Recombinant protein expression in Escherichia coli
ADVANTAGES
• Inexpensive setup and running costs
• High recombinant protein production levels
• Short timeline from cloning to protein recovery (1 week)
• Limited technical knowledge required for culturing
• Scalability from small (2 mL) to very large culture (>10,000 L) volumes
DRAWBACKS
• Inability to perform post-translation modifications (PTMs)
• Limited formation of disulphide bond
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Protein expression in E. coli: a plasmid backbone
• pET
• pGEX
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Co-expression of protein complexes in E. coli
• Example of the concatenation of two
vectors of the pET-MCN vector series:
pnEA-His (vector encoding an N-terminal
poly-histidine tag in front of protein A) and
pnCS (vector expressing the native
protein B).
• The pnEA-His (acceptor vector) is
linearized by removing with the restriction
enzymes BglII and SpeI part of its
promoter (T7 promoter and lacO).
• The full promoter of the pnCS (donor
vector) is cut out with the restriction
enzymes BglII and XbaI. After ligation of
the pnCS promoter with the linearized
pnEA-His vector, a new vector is obtained
based on the backbone of the pnEA-His
vector and whose promoter controls the
genes of proteins A and B.
The functional units within cells are often macromolecular complexes rather than single
species. Production of these complexes as assembled homogenous samples is a prerequisite
for their biophysical and structural characterization and hence an understanding of their
function in molecular terms. Co-expression in Escherichia coli can decipher the subunit
composition, assembly, and production of whole protein complexes.
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Wide range of E. coli strains
Strain Features
BL21 (DE3)
Most common host strain, enables high-level recombinant protein
expression
BL21 (DE3) pLysS
Enables high-level expression and suppression of T7 RNA
Polymerase basal level expression
BL21-CodonPlus
Improved expression of genes with codons rarely used in
bacteria
Rosetta
Improved expression of genes with codons rarely used in
bacteria
Arctic Express
Contains a plasmid encoding chaperonins to aid in folding and
allow expression at low temperature (12°C)
Shuffle T7 Express
Expresses DsbC to enable cytoplasmic disulphide bond
formation
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Expression in E. coli: tips and tricks
• Growth temperature: Typically 37°C, but lowering (25°C or 15°C) improves folding/solubility
• Expression with a fusion tag: polyhistidenes (6xHis, 10xHis or 12xHis)
Thioredoxin (Thx)
Maltose-binding protein (MBP)
Glutathione S-transferase (GST)
Small ubiquitin-like modifier (SUMO)
• Co-expression: Chaperonins (folding) and foldases (disulphide isomerase, DsbC)
Co-expression with interaction partners (co-solubility effects)
• Media: LB Broth, 2xLB Broth (simple and cheap), Terrific Broth (TB), 2YT
• Antibiotics: Selection of recombinant clones and prevention of contamination
Concentration of antibiotics in large-scale expression is decreased
• Codon optimization: Modifying codons in a gene sequence to match the codon
usage bias of the host cell used for expression
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Expression in yeasts
Concepts
Methods
Applications
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Expression in yeasts
• The yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris,
constitute another microbial systems in heterologous protein expression.
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Expression in Pichia pastoris
• Pichia pastoris (P. pastoris) is a widely
used protein expression host for the
production of biopharmaceuticals and
industrial enzymes
• P. pastoris belongs to methylotrophic
yeasts, which share a common pathway
to metabolize one-carbon compounds as
carbon and energy sources
• Vectors: The promoters for protein
expression in P. pastoris include inducible
promoters (AOX1, FLD1, ADH1, etc.) and
constitutive promoters (GAP, TEF1, etc.).
For P. pastoris, HIS4 (auxotrophic
markers) and zeocin resistance
(dominant markers) are the most
popularly markers used. The
heterologous proteins can be intracellular
or secreted expression. P. pastorishas
the ability to secrete high titres of proteins
into culture media
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Expression in insect cells
Concepts
Methods
Applications
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Production of proteins in insect cells
• Insect cells can efficiently express recombinant biologically active proteins and are mostly
used for the development of virus-like particles and vaccines. It has been proven that
insect cells are excellent platforms for the production of recombinant antibodies
• There are mainly three insect expression systems: baculovirus expression vector system
(BEVS), InsectSelect (IS) system and Drosophila expression system (DES)
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Introduction to baculovirus biology
Slack and Arif (2007)
• Insect-infecting enveloped DNA
viruses
• Baculoviruses are a very diverse
group of viruses with doublestranded,
circular, supercoiled
genomes, with sizes varying from
about 80 to over 180 kb, that encode
between 90 and 180 genes
• The genome is packaged in rodshaped
nucleocapsids that are 230-
385 nm in length and 40–60 nm in
diameter
• In the most well characterized
baculoviruses, the virions are present
as two types, occluded virions (ODV)
and budded virions (BV).
• Although these two types of virions
are similar in their nucleocapsid
structure, they differ in the origin and
composition of their envelopes and
their roles in the virus life cycle
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The baculovirus genome
• Circular, supercoiled dsDNA
genomes, with sizes varying from
about 80 to over 180 kb, that encode
between 90 and 180 genes
• The genome is packaged in rodshaped
nucleocapsids that are 230-
385 nm in length and 40–60 nm in
diameter
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Baculovirus morphogenesis: two types of virions
Circular dsDNA genome and associated proteins Circular dsDNA genome and associated proteins
Capsid shell (VP39, etc.) Capsid shell (VP39, etc.)
BV envelope (GP64, F) ODV envelope (P74, ODV-E66, PIFs etc)
Tegument (GP41), occlusion body (Polyhedrin)
Budded virus (BV) Occlusion-derived virus (ODV)
SlackandArif(2007)
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The structure of baculovirus nucleocapsid
Ohkawa et al. (2010)
J. Cell Biol.
Dr. Taro Ohkawa
University of California
Berkeley, USA
• Rod-like capsid (VP39, etc.)
• Distinct apical and basal structures
• Circular dsDNA genome
• PP78/83: Wiskott-Aldrich
syndrome protein (WASP)-like
protein
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Time-course baculovirus infection
Slack and Arif (2007)
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Baculovirus Expression Vector System (BEVS)
• Huge quantities of polyhedrin
produced by baculoviruses are the
fundamental features to develop the
viruses as vectors for foreign protein
production
• Later findings manifested that the
polyhedrin protein is not necessary for
baculovirus replication in infected
insect cells
• So, the strategy for expressing
recombinant protein by replacing viral
DNA sequence encoding polyhedrin
with the gene of interest is produced
• This theory became a reality when
scientists performed homologous
recombination of a polyhedrin region
in the viral genome with a plasmid
containing a foreign gene regulated by
the polyhedrin promoter to produce a
recombinant baculovirus, therefore
triggering the birth of baculovirusinsect
expression system
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Workflow of protein production using BEVS
https://www.intechopen.com/books/new-insights-into-cell-culture-technology/process-optimization-for-recombinant-protein-expression-in-insect-cells
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The MultiBac, a multi-expression system
• MultiBac consists of a baculoviral genome
optimized for multigene delivery and protein
complex expression (left).
• The genome is present as a bacterial artificial
chromosome (BAC) in E. coli cells supplying the
Tn7 transposase.
• Expression cassettes are assembled into the
multi-gene expression constructs and inserted
into the MultiBac genome by Tn7 transposition.
• A second entry option into the viral backbone is
provided distal from the Tn7 site, relying on Cre
recombinase catalysed site-specific integration
into a LoxP sequence (circle filled in red).
• Composite MultiBac baculoviral DNA containing
all DNA elements of choice is extracted from E.
coli cultures, followed by transfection into insect
cell cultures to manufacture functional MultiBac
virions.
• These are then used for a wide range of
applications (right), by the infection of insect cell
cultures or transduction of mammalian cells,
tissues and organisms.
Prof. Imre Berger
School of Biochemistry & Bristol Synthetic
Biology Centre, University of Bristol, UK
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Application of MultiBac system to large complexes
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The MultiBac system: successful structural stories
• Chromatin remodeling
enzymes SWR1 (14
subunits) and INO80 (11
subunits)
• The yeast polII DSIF-PAFSpt6
cryo-EM structure
• The CENPN-CENPAnucleosome
complex
• The human CPSF-160–
WDR33–CPSF-30–PAS
RNA quaternary complex
• The E9 polymerase
• The USP18-ISG15
complex
• The Separase-Securin
complex
• The cohesion loader Scc
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The MultiBac: a factory for synthetic virus-like particles
• A plasmid module comprising expression
cassettes for the capsid-forming influenza
H1N1 M1 protein (colored in grey) and a
fluorescent protein marker, mCherry
(colored in red), was introduced into the
MultiBac baculoviral genome by Cre
recombinase enzyme mediated plasmid
fusion into the LoxP site (circle filled in red,
gradient)
• Co-expression of HA, NA and M1 yields
synthetic influenza virus-like particles
(VLPs) resembling live influenza virus
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Selected vaccines produced by BEVS
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Baculovirus-mediated production of gene therapy vectors
• In 2002, Prof. Robert Kotin and
colleagues at the US National Heart,
Lung, and Blood Institute first
demonstrated its suitability for AAV
manufacturing
• They infected Sf9 cell lines — derived
from the fall armyworm — with three
different baculoviruses: two containing
essential genes for AAV particle
production (rep and cap), and one
containing the transgene sequence
intended for delivery
• In this manufacturing process, the
baculoviruses play a dual role,
functioning as the ‘helper’ virus normally
required for replication, as well as the
vehicle for AAV genetic material
• In their initial demonstration, Kotin’s
team achieved levels of productivity
comparable with existing AAV
manufacturing approaches — on the
order of 50,000 functional viral particles
per cell
Baculovirus-mediated production of adeno-associated
viral (AAV) vectors as gene therapy vehicles
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Development of AAV-based gene therapy products
• In 2017, FDA-approval of the first gene therapy product targeting a disease caused
by mutations in a single gene
• This product, LUXTURNA™ (voretigene neparvovec-rzyl; Spark Therapeutics, Inc.,
Philadelphia, PA), delivers a normal copy of the RPE65 gene to retinal cells for the
treatment of RPE65 mutation–associated retinal dystrophy, a blinding disease
• Many additional gene therapy programs targeting both inherited retinal diseases
and other ocular diseases are in development
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World's most efficient large-scale AAV manufacturing
• Virovek has developed a patented BAC-toAAV
technology that utilizes the baculovirus
expression system to produce AAV vectors in
insect cells under serum-free condition
• The capability to generate over 3e+16vg of
AAV vectors with a single production run,
which is unmatched by any other AAV
production system
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Applications of BEVS
• Nowadays, baculovirus-insect expression
system has been relatively mature in many
labs for protein production and is becoming a
leading method to produce high quality
proteins among the eukaryotic expression
systems
• The use of BEVS to express the protein of
interest requires two steps. In the first step,
the insect cells are cultured to the desired
concentration. In the second step, the
baculovirus is applied to infect the insect cells.
The virus that infects host cells will dominate
the gene expression mechanism in the host
cell and trigger the production process of the
target protein
• Yet, several mature expressing vectors have
been developed, including Autographa
californica multiple nucleopolyhedrovirus
(AcMNPV) that is the most extensively used
• The ability to produce biologically active
mammalian proteins makes the BEVS a
powerful tool over yeast and bacterial
expression systems
Overview of the various applications of the
baculovirus expression system.
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Applications of baculovirus technologies
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Advantages and drawbacks of baculovirus expression
ADVANTAGES
• Recombinant protein has complete biological function, such as protein
correct folding and disulfide bond
• Post-translation modification (glycosylation etc.)
• High level of expression, up to 50% of the total protein amount
• Accommodate large insert protein
• Simultaneously express multiple genes (multi-expression)
• In general, it is safer to use than mammalian virus, since it has limited host
range and does not infect vertebrates
DRAWBACKS
• The drawback is that exogenous protein expression is under control of the
very late viral promoter, where the cells begin to die due to viral infection
• Insect expression system is normally used for production of membrane
proteins, although the glycosylations may be different from those found in
vertebrates
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Virus-free expression in insect cells
• Overview of the general procedure to produce
stably transformed D. melanogaster S2 cell
lines for recombinant protein expression
• Protein expression can be initiated at different
points, starting with transient expression
immediately after transfection followed by
stable expression in a polyclonal cell line and
finally the selection of a highly productive
monoclonal cell line
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The different plasmid sets to generate stable S2 cell lines
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Expression in mammalian cells
Concepts
Methods
Applications
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Mammalian expression system
• Recombinant proteins expressed in mammalian cell commonly use plasmid transfection
and viral vector infection. Stable cell line using plasmid transfection takes several weeks
or even months, while the virus vector can quickly infect cells within a few days
• Depending on the temporal and spatial differences in protein expression, the expression
system can be divided into transient, stable and induced expression systems
• Transient expression system refers that host cell cultures without selection pressure and
exogenous vector gradually lost while cell division. The target protein expression duration
is short. The advantage of transient expression system is simple and short experimental
period
• Stable expression system means that the carrier DNA replicate and express long time
stably in host cell. Due to the need of select resistance and pressure steps, stable
expression is relatively time-consuming and laborious
• Induction expression system refers that the target gene begins to express when induced
by foreign small molecules. The use of heterologous promoters, enhancers and
amplifiable genetic markers can increase protein production
• The mammalian expression system has unique advantage in protein initiation signals,
processing, secretion, glycosylation and is suitable for expressing intact macromolecules
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Mammalian stable cell line generation
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Expression in Leishmania tarentolae
Concepts
Methods
Applications
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LEXSY - Eukaryotic protein expression in L. tarentolae
The unicellular kinetoplast protozoan Leishmania tarentolae, isolated from the Moorish
gecko Tarentola mauritanica, not pathogenic to mammalians (Biosafety level 1) – was
turned into the protein-producing host of our eukaryotic protein expression system LEXSY:
• eukaryotic host as easy to handle as E. coli: no specific labware, no cell
biology equipment required
• fully eukaryotic protein expression machinery with post-translational
modifications, including glycosylation and disulfide bond formation
• shuttle vectors: cloning in E. coli, expression in LEXSY host
• constitutive or inducible, intracellular or secretory expression of target proteins
• stable expression strains for constant protein production
https://www.jenabioscience.com/lexsy-expression
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Leishmania tarentolae: a little-known expression host
• Leishmania tarentolae, which can be easily
cultured and genetically manipulated, is
harmless (unless you are a gecko from
Africa)
• It is closely related to other kinetoplastida,
including important human pathogens such
as L. donovani (causing kala azar),
Trypanosoma brucei (sleeping sickness)
and T. cruzi (Chagas disease)
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Emerging new expression tools
Concepts
Methods
Applications
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The CRISPR/Cas9 system
• The Cas9 (CRISPR associated protein 9) is a protein which plays a vital role in the
immunological defense of bacteria against DNA viruses, and which is used in genetic
engineering. Its main function is to cut DNA and therefore it can alter a cell's genome
• Structurally, Cas9 is an RNA-guided DNA endonuclease enzyme associated with
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity
system in Streptococcus pyogenes
• Cas9 performs this by unwinding foreign
DNA and checking for sites complementary
to the 20 bp spacer region of the guide RNA
• If the DNA substrate is complementary
to the guide RNA, Cas9 cleaves
the invading DNA
Crystal structure of S. pyogenes Cas9 in complex
with sgRNA and its target DNA at 2.5 Å resolution,
Nishimazu et al., Cell 156: 935–49 (2014)
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The CRISPR/Cas9 system on YouTube
https://www.youtube.com/watch?v=bXnWIk8FgKc
https://www.youtube.com/watch?v=OjNrbPMXyMA
https://www.youtube.com/watch?v=0dRT7slyGhs
https://www.youtube.com/watch?v=2pp17E4E-O8
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The CRISPR/Cas9 system: key elements
• Cas9 nuclease specifically cleaves double-stranded
DNA activating double-strand break repair
machinery
• In the absence of a homologous repair template
non-homologous end joining can result in indels
disrupting the target sequence
• Alternatively, precise mutations and knock-ins can
be made by providing a homologous repair template
and exploiting the homology directed repair pathway
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Cell-Free Protein Synthesis (CFPS)
• Cell-free protein synthesis
(CFPS) is a platform technology
that provides new opportunities
for protein expression
• The advantages of CFPS over in
vivo protein expression include
its open system, the elimination
of reliance on living cells, and the
ability to focus all system energy
on production of the protein of
interest
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Cell-free platforms and their applications
(A) Web of the applications enabled by low adoption cell-free platforms. Connections shown
are based on applications that have been published or that have been proposed in
publications.
(B) Cumulative number of peer-reviewed publications over the last 60 years for cell-free
platforms.
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Preparation of cell-free extract and set-up of CFPS reactions
General workflow for preparation of cell-free extract and set up of CFPS reactions.
A visualization from cell growth to the CFPS reaction is depicted above for a new
user, highlighting the main steps involved.
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Comparison of CFPS platforms
A. Batch reactions contain all the necessary reactants within a single reaction vessel.
B. Continuous exchange formats utilize a dialysis membrane that allows reactants to move
into the reaction and byproducts to move out, while the protein of interest remains in the
reaction compartment.
C. Continuous flow formats allow a feed solution to be continuously pumped into the reaction
chamber while the protein of interest and other reaction byproducts are filtered out of the
reaction.
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CFPS reaction incorporating nonstandard amino acids
• Scheme of cell-free protein
synthesis reaction
incorporating nonstandard
amino acid to investigate
the effect of RF1
• Cell extracts containing
transcription and translation
machinery are prepared
from rEc.E13 or
rEc.E13.ΔprfA strains
• Plasmid DNA template of
sfGFP containing single or
multiple amber codon
sites, orthogonal
tRNA/aaRS, NSAA, T7
RNA polymerase, and other
cofactors are added as
necessary to activate the
cell-free protein synthesis
(CFPS) reaction
p-acetyl-L-phenylalanine
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CFPS from extracts of a genomically recoded organism
• Schematic of the production and utilization of crude extract from genomically recoded organisms with
plasmid overexpression of orthogonal translation components for cell-free protein synthesis
• CFPS reactions are supplemented with the necessary substrates (e.g., amino acids, NTPs, etc.) required
for in vitro transcription and translation as well as purified orthogonal translation system (OTS) components
to help increase the ncAA incorporation efficiency
• aaRS, aminoacyl tRNA synthetase; ncAA, non-canonical amino acid; T7P, T7 RNA polymerase; UAG,
amber codon
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Protein purification methods
Concepts
Methods
Applications
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Recombinant protein purification: step by step
The aim of a purification procedure is to obtain a highly pure and stable protein at
an appropriate concentration in a buffer compatible with the intended application.
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Chromatography columns in protein purification
Technique Stage Description
Affinity Chromatography
(AC)
Capture or
Intermediate
Based on a reversible interaction between the
protein/affinity tag and a specific ligand
Ion Exchange
Chromatography (IEX)
Capture or
Intermediate
Separates proteins based on their net surface charge
Hydrophobic Interaction
Chromatography (HIC)
Intermediate
Binding under high salt conditions, generally performed
following an ammonium sulphate precipitation step
Size Exclusion
Chromatography (SEC)
Polishing
Separates proteins based on their hydrodynamic volume
(size)
Reverse Phase
Chromatography (RPC)
High-resolution
chromatography based on weak
hydrophobic interactions. Harsh conditions generally only
suitable for purification of peptides
Chromatography is the most powerful and commonly used means of purifying recombinant
proteins. Each technique separates proteins based on different properties, so it is often
advantageous to combine several types to maximise separation of the recombinant protein
from host cell proteins.
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Affinity chromatography: fusion tags
Fusion tag Function
Size
(kDa)
Description
Polyhistidine (e.g.
6xHis, 10xHis)
Affinity 1-2
The most commonly used affinity tag, binds
to metal ions
Strep-tag II Affinity 1 High affinity for engineered streptavidin
Thioredoxin (Trx) Solubility 12
Aids in refolding proteins that require a
reducing environment
Small Ubiquitin-like
Modifier (SUMO)
Solubility 12
Contains a native cleavage sequence
enabling tag removal with SUMO protease
Glutathione Stransferase
(GST)
Solubility, affinity 26
High affinity for glutathione, often needs to
be removed due to large size
Maltose Binding Protein
(MBP)
Solubility, affinity 41
Binds to maltose, often needs to be
removed due to large size
Fusion tags can improve protein expression, stability, resistance to proteolytic
degradation and solubility.
• Fusion tag orientation (N- or C-terminus)
• Combinatorial fusion tags (Trx/GST/MBP with an affinity tag, e.g. 6xHis)
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Variety of proteases for fusion tag removal
• Human Rhinovirus (HRV 3C)
• PreScission protease
• Tobacco Etch Virus (TEVp)
• SUMOp
• Thrombin
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Column chromatography instrumentation
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Protein characterization: an aggregation problem
A key challenge in recombinant protein production is to maintain and store the target
protein in a soluble and stable form. Protein aggregation can compromise protein function
and thus it is necessary to overcome this challenge to generate functionally active protein.
Detection of protein aggregation
• Analytical size-exclusion chromatography (SEC)
• Dynamic light scattering (DLS)
• Analytical ultracentrifugation (AUC)
Troubleshooting
• Culture conditions (e.g. reducing temperature)
• Buffer composition (ionic strenght, pH, reducing agents)
• Presence co-factors (Acetyl-CoA, metal ions)
• Fusion tags (Trx, MBP, SUMO)
• Minimising sample handling
• Avoiding time delays between purification steps
• Performing purification steps at 4°C
• Store purified proteins in -80°C
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Protein quality control (QC) analyses
High purity and homogeneity of the protein sample are crucial for the
downstream processes to be successful.
• Dynamic light scattering (DLS): To characterize the polydispersity of sample
Identification of different oligomeric forms or
aggregates, which are preventing crystallization
• Differential scanning fluorimetry (DSF): analysis of protein stability
To characterize the stability of the protein in different
buffers and in the presence of different ligands, which
stabilize the protein for crystallization
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Success stories
66
Development of expression and purification protocol
for Schistosoma mansoni HDAC8: mini-scale tests
67
Large-scale production and crystallization of smHDAC8
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Conclusions
• The project design is ultimately determined by the
end-use of the recombinant protein
• The overall success of a project lies in an effective
project design
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Questions
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Molekulární biotechnologie cvičení
Středa 9. říjen 2019 10:00 – 11:50: A13/332
Středa 9. říjen 2019 14:00 – 15:50: A36/308
72
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
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Baculovirus biology
• Extracellular enveloped baculovirus virions
can be found in two forms: OV (occluded
virus) and BV (budded virus). The
nucleocapsid is about 21 nm x 260 nm.
• Circular dsDNA genome , 80-180 kb in
length, encoding for 100 to 180 proteins.
BACULOVIRUS INFECTION CYCLE
• Attachment of the viral glycoproteins to host receptors mediates endocytosis of the virus into the host
cell.
• Fusion with the plasma membrane.
• The DNA genome is released into the host nucleus.
• Immediate early phase: host RNA polymerase transcribes viral genes involved in the regulation of the
replication cascade, prevention of host responses and viral DNA synthesis.
• Late phase: The virally encoded RNA polymerase expresses late genes.
• Replication of the genome by rolling circle in nuclear viral factories.
• Nucleocapsids are formed which can either bud out through the cellular membrane and disseminate the
infection or be occluded for horizontal transmission.
• Occlusion phase: the virus becomes occluded in the protein polyhedrin and the polyhedral envelope
(calyx) is produced.
• Lysis of the cell releases the occluded virus.
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The replication cycle of baculovirus
• Baculovirus is a member of the insect-borne virus family and was originally used as a biopesticide with a
large double-stranded circular DNA genome. The virus is enveloped, rod-shaped particles, ranging from 30
to 60 nm in diameter and from 250 to 300 nm in length.
• The natural hosts for these viruses are insects, and Autographa californica (AcMNPV ) is the most studied
member of the family, which was developed into a recombinant baculovirus vector and is still under
application today.
• AcMNPV and other baculoviruses are able to generate ‘‘polyhedra’’ or ‘‘occlusion bodies’’ in the infected
cells of insects. In the late phase of infection, dozens of polyhedra are formed in the host cell nuclei, which
accommodate progeny virions encased by a protective paracrystalline array comprised of virus-encoded
protein-polyhedrin.
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A comparison of BEVS processes with low and high MOI
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How can baculoviruses express
very late genes at such high levels?
There is no evidence that the baculovirus RNA polymerase has an intrinsic ability for high level gene
expression. In fact, it may not bind to the very late promoter region with high levels of tenacity as
reflected in the fact that the footprint of the polymerase on genomic DNA has never been reported
despite an extensive effort by at least one laboratory.
It is likely that high levels of gene expression are influenced by several features of baculovirus biology.
These include: i) the amplification of genes by DNA replication; ii) the shutoff of most late transcription,
possibly by DNA binding proteins that coat the DNA and thereby make RNA polymerase available for
very late transcription; iii) the efficiency of the late polymerase and VLF-1 in recognizing and initiating
from very late promoter elements; iv) the efficiency of LEF-4 in capping the mRNA a possible role for
LEF-2 and PK-1. As mentioned above, the 5' untranslated region of p10 mRNA appears to be capable
of facilitating cap-independent translation, which may reduce the reliance of these transcripts on LEF-4
activity. Other factors that might enhance translation of very late expressed mRNAs have not been
identified.
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Baculovirus-mediated production of AAV vectors
Gene transfer and gene therapy are powerful approaches for many biological research
applications and promising avenues for the treatment of many genetic or cancer diseases.
The most efficient gene transfer tools are currently derived from viruses. Among them, the
recombinant adeno-associated viruses (AAVs) are vectors of choice for many
fundamental and therapeutic applications. The increasing number of clinical trials involving
AAVs demonstrates the need to implement production and purification processes to meet
the quantitative and qualitative demands of regulatory agencies for the use of these vectors
in clinical trials. In this context, the rise of production levels on an industrial scale appeared
essential. The introduction, in 2002, of an AAV process using a baculovirus expression
vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in
expanding the AAV production in insect cells has been to switch the process to bioreactor
systems, which are the ideal equipment for scaling up. We describe here a method for
producing AAV vectors using the BEVS which can be easily used by research laboratories
wishing to overcome the difficulties associated with the scaling up of production levels. The
method provides sufficient quantities of AAV vectors to initiate preclinical projects in large
animal models or for research projects where a single batch of vectors will consolidate the
repeatability and reproducibility of in vitro and especially in vivo experimental approaches.
Methods Mol. Biol. 2019; 1937: 91-99. doi: 10.1007/978-1-4939-9065-8_5