Recombinant proteins

Tema 6
Ismael Mingarro (UVEG)

Tecnologia de Proteïnes
Grau en Biotecnologia

Recombinant • 1. Cloning strategies

– 1.1 Cloning by restriction/ligation
protein expression – 1.2 Synthetic genes
& purification – 1.3 Cloning without restriction/ligation
• 1.3.1 Gateway technology
• 1.3.2 In Fusion/Creator technology
• 1.3.3 Topo technology
• 1.3.4 LIC technology
• 2. Expression systems
– 2.1 Prokaryotic
– 2.2 Eukaryotic
• 2.2.1 Yeast
• 2.2.2 Baculovirus/insect cells
• 2.2.3 Mammalian
– 2.3 Cell-free
• 3. Protein solubilization and
Ismael Mingarro (UVEG)

renaturalization
– 3.1 Refolding of inclusion bodies
– 3.2 Chaperone co-expression
– 3.3 Critical factors and solutions
 Why bother with recombinant fusion
protein expression?

1.- for efficient and selective purification

2.- to minimize proteolysis
3.- to optimize translation efficiency
4.- to suit specific scenarios: antibody
production, biochemical experiments, structural
Ismael Mingarro (UVEG)

biology, industrial (biotechnological) use,…

How much?

Purpose Yield required

Study protein function ng-μg

Generate antibodies mg
Protein structure 10-100 mg
Diagnosis or therapy mg-g
Ismael Mingarro (UVEG)

Biotechonogical applications >g

 1. Cloning Strategies

1.1 Cloning by restriction/ligation

1.2 Synthetic genes
1.3 Cloning without restriction/ligation:
1.3.1. Gateway technology
1.3.2. In Fusion / Creator technology
1.3.3. TOPO technology
1.3.4. LIC technology
Ismael Mingarro (UVEG)

Stanley Cohen Herbert Boyer

1. Cloning Strategies
1.1 Cloning by restriction/ligation
Boyer’s Lab
isolated an enzyme
that could be used
to cut strings of
DNA into precise
and "cohesive"
segments
Cohen´s Lab
developed a method to
introduce antibiotic-
carrying plasmids into
certain bacteria
Ismael Mingarro (UVEG)

6
 1. Cloning Strategies
1.2 Synthetic genes
Ismael Mingarro (UVEG)

1. Cloning Strategies
1.3 Cloning without restriction/ligation:
1.3.1. Gateway technology
1.3.2. In Fusion / Creator technology
1.3.3. TOPO technology
1.3.4. LIC technology
In-Fusion enable
direccional, seamless
cloning of any PCR
fragment (or multiple
fragments) into any
linearized vector in a
single 15-30 minute
reaction.
Ismael Mingarro (UVEG)

8
 2.- Expression systems

2.1- Prokaryotic system: Escherichia coli

2.2- Eukaryotic systems:

2.2.1. Yeast: Saccharomyces cerevisiae,
Pichia pastoris, P. methanolica,…
2.2.2. Baculovirus/insect cells
2.2.3. Viral/mammalian cells

2.3- In vitro systems

Ismael Mingarro (UVEG)

Expression systems

EASY DIFFICULT
USE
LOW HIGH
COST

Prokaryotic
Yeast Mammalian
Insect
Ismael Mingarro (UVEG)

- +
POST-TRANSLATIONAL
MODIFICATIONS
Ismael Mingarro (UVEG)

Develop a strategy that fits your protein…

The perfect system E. coli Yeast Insect

- non-pathogenic -yes -yes -yes

- fast growth ~20 minutes ~2 hours ~24 hours
- microbial system -yes -yes -no
- inexpensive -yes -yes -no
- high expression level -up to g/L -mg/L -mg/L
- regulatable -yes -yes -yes
- properly modified protein -minimal -some -much
- easy to purify product -possibly -easy -easy
Ismael Mingarro (UVEG)

…and your money!!!

 2.1- Prokaryotic systems: E. coli
- selectable marker: maintenance of the vector in the cell
Expression vector elements
– bla, encoding β-lactamase: ampr
– cat, encoding chloramphenicol acetyltransferase: chlor
– tet, encoding a membrane associated protein, tetr, that prevents the antibiotic from entering the cell
- origin of replication: control of the plasmid replication as an independent extrachromosomal
element
– ColE1/pBR322 origin
– pUC origin
- translation initiation sequence: RBS (ribosome binding site, Shine-Dalgarno)
- promoter: inducible production of large amounts of mRNA
– lac promoter (tac, trc): control elements from the E. coli β-galactosidase gene; induction with IPTG, a
lactose analog that binds to the lac repressor blocking it
– pL promoter: promoter and control elements of bacteriophage lambda; induction by a thermal shift that
denatures cI repressor
– T7 RNA polymerase promoter: induction of T7 polymerase synthesis which is under the control of an
inducible promoter such as lac (T7 promoter / lac operator)
Ismael Mingarro (UVEG)

target gene

E. coli compartments for protein production

Periplasm

Cytosol
Ismael Mingarro (UVEG)

Extracellular
 Control elements of the pET system (T7lac)

targe
t gen
e

s
ue
tiq
àc
Pr
Ismael Mingarro (UVEG)

Uninduced state: T7 RNA polymerase under the

control of the lac promoter: some degree of
transcription but inhibited by T7 lysozyme and presence of
a lac operator downstream from the T7 promoter

Control elements of the pET system (T7lac)

IPTG induction

targe
t gen
e
s
ue
tiq
àc
Pr
Ismael Mingarro (UVEG)

Induced state: IPTG binds to the lac repressor and

prohibits its binding to the lac operator. T7 RNA
polymerase is transcribed, binds to the T7 promoter and
the gene of interest is expressed
 Strain Company Main feature
E. coli strains
BL21(DE3) Novagen protease deficient

BL21(DE3) pLysS Novagen produce lysozyme

BL21(DE3) pLysE Novagen produce more lysozyme than S

Tuner(DE3) pLysS Novagen lac permease deletion mutant

OrigamiB(DE3)pLysS Novagen trx and glutathione reductase mutant > disulfide bridges

Rosetta(DE3)pLysS Novagen supply tRNA for 6 rare codons

C41(DE3) Avidis SA tolerant to toxic and membrane proteins

C43(DE3) Avidis SA idem for one specific protein

B834(DE3) Novagen met auxotroph > Se-Met labelling

Ismael Mingarro (UVEG)

Electron micrographs of thin sections of E.

coli C43(DE3) cells over-producing subunit
β of E. coli ATP synthase, 3 and 18 h after
induction at 37 or 25°C, respectively.

2.2.1- Eukaryotic syst.: Yeast (Pichia pastoris)

- eukaryote as easy to manipulate as E. coli Cloning in Pichia 2-3 days
expression vector
- easier, faster, less expensive than other eukaryotes
- same genetic, biochemical and molecular biology Linearization by 1 hour
techniques than for S. cerevisiae restriction enzyme

- post-translational modifications Transformation in 2 hours

P. pastoris strain
- high biomass
Select clones on
medium with 3-4 days
Zeocyn or without
Histidine

- generally high expression levels Small scale

expression on 10-20 3-4 days
colonies
- methylotrophic yeast: can metabolize methanol as its
sole source of carbon with alcohol oxidase
Ismael Mingarro (UVEG)

Total time = 3 weeks

- promoter of the alcohol oxidase 1 (AOX1) used to drive the expression of the
gene of interest
- efficient secretion into the medium of protein fused to the α-factor or a native
secretion signal > very low level of native yeast secreted proteins (easier to purify)
 P. pastoris strains
Strain Genotype Phenotype
X33 wild type Mut+
GS115* his4 His- Mut+
KM71H° arg4 aox1::ARG4 MutS Arg+

KM71*° arg4 his4 aox1::ARG4 His- MutS Arg+

* Strains with a mutation in the histidinol deshydrogenase gene (his4), which

prevents them from synthesizing histidine; expression plasmids carrying the
HIS4 gene complement his4 in the host and so transformants are selected for
their ability to grow on histidine-deficient medium.
° Strains with a mutated arginosuccinate lyase gene (arg4) and with their wild-
type AOX1 gene replaced by the wild-type ARG4 gene.
Mut+: methanol utilization plus
Wild type ability of strains to metabolize methanol as the sole source of carbon
Ismael Mingarro (UVEG)

MutS: methanol utilization slow

Phenotype caused by the loss of alcohol oxidase activity encoded by the AOX1
gene but presence of the wild-type AOX2 resulting in a slow growth phenotype
on methanol medium

2.2.2 Eukaryotic systems: Baculovirus/Insect cells

- rod-shape insect virus (450 nm in length and 45 nm in

diameter) with a circular double-stranded DNA
- life cycle divided in 3 main stages: early, late and very late
phases
- expression of recombinant proteins during the very late
phase under the control of the promoter of polyhedrin or P10
- proper protein folding and post-translational modifications
- intracellular or secreted expression
- generally high expression levels
Ismael Mingarro (UVEG)

- wide range of commercially available plasmids

Disadvantage: higher cost than for bacterial and yeast
expression systems
 Phase I
Baculovirus
expression

Phase II

II
eI
Ismael Mingarro (UVEG)

as
Ph

21

2.2.3 Eukaryotic systems: Mammalian cells

RheoSwitch® Mammalian Inducible Expression System. (New England BioLabs)

RheoSwitch Ligand (RLS1)

Ismael Mingarro (UVEG)

immunoblots!
 2.3- In vitro systems: Cell-free protein synthesis

- protein biosynthesis does not require the integrity of

the cell and can continue after the cell disruption
Ismael Mingarro (UVEG)

- in a mRNA-free cell extract, a DNA species (plasmid,

isolated gene or synthetic DNA fragment) could be
transcribed into a mRNA, that could be translated into
the corresponding protein

RTS Technologies…

RTS continuous-exchange cell-freetechnology (CECF) increases

yields considerably

home
made:
Ismael Mingarro (UVEG)
 3. Protein solubilization:
3.1 Refolding of inclusion bodies

Centrifugation
Bacterial 30’ at 15000 g SDS-PAGE
lysate Supernatant
(S)
P Mw S

Membranes
Inclusion bodies Pellet (P)
sh
wa a and 100
ure X-
M on
1-4 Trit
- 5 %
0.5

N-terminal or C-terminal 6His tag

Solubilization in 6M
guanidinium
Centrifugation chloride or 8M urea
Ismael Mingarro (UVEG)

30’ at 22000 g
(x3)
Ni2+ affinity column in
denaturing conditions
(8M urea)

Inclusion bodies

Ni2+ affinity purification

His-tag

Binding

Ni-sepharose Elution with

250 mM
Wash imidazole

(0-20 mM
imidazole)

Pure soluble protein at

high concentration but
denaturated in Refolding
Ismael Mingarro (UVEG)

50 mM Tris pH 7.5,150
mM NaCl, 8M urea, 250
mM imidazole
 Refolding
Theory:
Progressive or instantaneous removing of the chaotropic agent (urea,
guanidinium chloride…) until its solubilizing power is canceled out.
If the protein is in a native form: -> soluble form
In the contrary: -> precipitation
Different refolding protocols:
- solid phase
- dialysis
- dilution Sans Titre

0.08
80.0

70.0
Example of refolding kinetics by
monitoring the formation of
0.06
60.0 insoluble aggregates at 340 nm
Abs340nm (UA)

50.0
ORF52
-3
Légende /10

0.04
40.0
Tris-HCl 50 mM pH 7.5
30.0 NaCl 200 mM
DTT 1mM
Ismael Mingarro (UVEG)

0.02
20.0

10.0
Tris-HCl 50 mM pH 7.5
0
0.0 NaCl 200 mM
DTT 1mM
-10.0
0.000 1.00 2.00 3.00
Arginine 800 mM
1 2 3

Time (min) after removing of the chaotropic agent

Légende

IB refolding screening using a 96-well format

The turbidity of a solution is assessed by measuring the optical density (OD) at
340 nm, before and after adding the protein. If the protein remains soluble, the
absorbance remains unchanged. In the opposite case, the OD increases
proportionally to the amount of precipitate produced.

96 buffers (fractional factorial approach):

REDUCING NDSB
pH (50mM) PRECIPITANTS ADDITIVES
AGENT (100mM)
Glycerol 20%
Na Acetate pH 4 Arginine
NaCl 100mM
MES pH 5 800mM
NaCl 200mM NDSB 195
MES pH 6 β-mercaptoethanol
KCl 100mM NDSB 201 Glucose
TRIS pH 7 10mM
EDTA 1mM NDSB 256 500mM
TRIS pH 8
PEG 4000 0.05%
CHES pH 9 Cocktail *
PEG 400 0.05%

* NADH, Thiamine, Biotine, CaCl2, MgCl2, CuSO4, ZnCl2, CoSO4, ADP, NiCl2, 50µM each
… and 16 buffers from the « Fold-It » screen (Hampton)
Ismael Mingarro (UVEG)

- 95 µL of each buffer
- 5 µL of protein (at 4mg/mL or more)
- mix and measure the OD at 340 nm: immediately, after 2 hours, next day

Vincentelli R. et al. (2004) Protein Sci. 13, 2782

 1 2 3 4 5 6 7 8 9 10 11 12
NaCl 200
KCl 100 βmercapt
A βmercapt βmercapt NaCl 100 βmercapt
PEG 400 βmercapt o KCl 100 PEG 4000
o o βmercapt Glycerol EDTA NDSB201 o
Glucose o NDSB201 NDSB195 Glucose
Arginine NDSB256 o NDSB195
NDSB256 Glucose
Arginine
Glycerol
B PEG 4000 EDTA PEG 400
βmercapt βmercapt
EDTA NaCl 100 βmercapt NaCl 200 βmercapt βmercapt βmercapt βmercapt
NaCl 200 NDSB256 o o
Arginine Arginine o NDSB195 o o o o
NDSB256 NDSB195
NDSB201 NDSB201 Arginine
Glucose
NaCl 200
C PEG 400 βmercapt
Glycerol
βmercapt
Glycerol
KCl 100 βmercapt o o PEG 400 NDSB256
Cocktail NDSB256 βmercapt KCl 100 Bmercapt EDTA
Glucose o NDSB195 NDSB201 NDSB256 Glucose
Arginine o o
NDSB201 Glucose Glucose Arginine

D PEG 400
NaCl 100
βmercapt Glycerol PEG 4000 βmercapt
βmercapt NaCl 200 NaCl 100 βmercapt
/ PEG 4000 o / KCl 100 βmercapt NDSB256 o
o NDSB201 NDSB201 o
NDSB256 o Arginine Arginine
NDSB195
Glucose
E KCl 100
EDTA
PEG 4000 EDTA Glycerol βmercapt
NDSB201 NDSB195 βmercapt PEG 4000
βmercapt Cocktail Arginine NDSB256 βmercapt o Glucose /
Arginine Glucose o NDSB195
o Glucose o NDSB201
NDSB195
Arginine
F
KCl 100
NaCl 100 NaCl 200 PEG 4000 NaCl 200
βmercapt βmercapt
βmercapt βmercapt βmercapt PEG 400 βmercapt βmercapt βmercapt NaCl 100 βmercapt
NDSB201 o o
o o o NDSB195 o o o NDSB256 o
NDSB201 NDSB195
NDSB256 Glucose Glucose NDSB256
G Glucose
EDTA
βmercato
Glycerol NaCl 100 βmercapt NDSB195
NDSB195 PEG 400 / Cocktail FoldIt N°1 FoldIt N°2 FoldIt N°3 FoldIt N°4
NDSB201 Glucose o Arginine
Arginine
H Arginine
Ismael Mingarro (UVEG)

FoldIt FoldIt FoldIt FoldIt FoldIt FoldIt FoldIt

FoldIt N°5 FoldIt N°6 FoldIt N°7 FoldIt N°8 FoldIt N°9
N°10 N°11 N°12 N°13 N°14 N°15 N°16

Na acetate pH 4 TRIS pH 7
MES pH 5 TRIS pH 8
MES pH 6 CHES pH 9

3. Protein solubilization:
3.2 Chaperone co-expression
What is a chaperone?

- upregulated upon heat shock or other insults that increase cellular protein
misfolding
- classified as stress or heat shock proteins (Hsps)
- relied on the differential exposure of structured hydrophobic domains to
the solvent to bind nonpolar segments that would normally be buried within
the core of their substrates

3 functional subclasses based on their mechanism of action:

- ’Folding’ chaperones: rely on ATP-driven conformational changes to
mediate the net refolding:unfolding of their substrates
- ’Holding’ chaperones: maintain partially folded proteins on their surface
Ismael Mingarro (UVEG)

to await availability of folding chaperones

- ’Disaggregating’ chaperones: promote the solubilization of aggregated
proteins
 Chaperone-assisted protein folding in the
cytoplasm of E. coli
– 1rst step: nascent polypeptides TF: trigger DnaK-DnaJ
encounter TF or DnaK-DnaJ which factor 1
engage solvent-exposed stretches of
hydrophobic amino acids (1)
– 2nd step: after undocking from DnaK 2a
mediated by GrpE, folding intermediate
may reach a native conformation (2a), Inclusion 2b
cycle back to DnaK-DnaJ (2b) or be bodies
stress
transferred to GroEL-GroES (2c)
3
– 3rd step: in times of stress, proteins 4 ss 2c
re
unfold and aggregate. DnaK-DnaJ and st
e
GroEL-GroES are assisted by holding a tiv
xid

s
es
chaperones that stabilize partially folded o

str
proteins without actively promoting their

al
rm
remodeling (3).

e
th
– 4th step: under severe or prolonged
Ismael Mingarro (UVEG)

stress conditions, a disaggregating

chaperone (ie. ClpB) promotes the
shearing and disaggregation of unfolded
proteins and cooperates with DnaK-
DnaJ-GroEL to reactivate them once
stress has abated (4) Baneyx F. and Mujacic M. (2004) Nature Biotech. 22:1399

Chaperone co-expression
- improvement
of the yields of
soluble proteins
in the cytoplasm
of E. coli
Ismael Mingarro (UVEG)

- availability of a number of plasmids compatible with the

routinely-derived expression vectors
Nishihara K. et al. (1998) Appl. Environ. Microbiol. 64:1694
 3.3 Summary E. coli expression:
Factors critical to solubility

soluble
protein

(Tema 5)
Ismael Mingarro (UVEG)

33

Strategies for
overcomming common
problems during
recombinant protein
expression in E. coli

Rosano et al. (2019)

Protein Sci. 8:1412
Ismael Mingarro (UVEG)

Rosano GL. & Ceccarelli EA. (2014)

Front. Microbiol. 5:172 34