Ribosome Display: In Vitro Selection of
Ribosome display is an in vitro
technology to identify
and evolve proteins or peptides binding to a given
target (Fig. 1) (Hanes et al.
, 2000a). While most selection
technologies need living cells to achieve the essential
coupling of genotype and phenotype, ribosome
display uses the ribosomal complexes formed during in vitro
translation to generate the physical coupling
between polypeptide (phenotype) and mRNA (genotype)
(Amstutz et al.
, 2001). Hence, no transformation
step limiting the size of the usable library is necessary,
allowing the selection from very large combinatorial
libraries. In addition, the rapid selection cycles require
an integral polymerase chain reaction (PCR) step,
which can be used for randomization, making this
method ideal for directed evolution experiments. The
fact that the ribosomal complex used for selection is
not covalent allows an uncomplicated separation of
the mRNA from the selected ribosomal complexes,
even if the selected molecules bind the target with very
high affinity or are even trapped covalently (Amstutz et al.
, 2002; Jermutus et al.
, 2001). All these benefits
make ribosome display a good alternative to other
selection techniques, such as phage display (Smith,
Ribosome display has been applied successfully for
the selection of peptides (Matsuura and Plückthun,
2003; Mattheakis et al.
, 1994), as well as folded proteins
such as antibody fragments (Hanes and Plfickthun,
1997; He and Taussig, 1997; Irving et al.
, 2001). Ribosome
display can also be considered for the screening
of cDNA libraries for interaction partners. Ribosome
display ultimately selects always for a specific binding
event. However, by designing the selection pressure
carefully, molecules can be selected for many other
parameters, such as enzymatic turnover (by selection
with a suicide inhibitor, or active site ligand) (Amstutz et al.
, 2002; Takahashi et al.
, 2002), protein stability
(by selecting for binding under conditions where
most library members will not fold) (Jermutus et al.
2001), or protein biophysical properties (resistance to
proteases and nonbinding to hydrophobic surfaces)
(Matsuura and Plfickthun, 2003). It is the combination
of this array of selection pressures with the
convenient PCR-based randomisation techniques that
makes ribosome display a powerful and versatile
II. MATERIALS AND
|FIGURE 1 Ribosome-display selection cycle. The DNA of the library of interest, fused in frame to a spacer
carrying no stop codon, is transcribed in vitro. The resulting mRNA is used for in vitro translation. After a
short time of translation (a few minutes) the ribosomes have probably run to the end of the mRNA and synthesized
the encoded protein, but because of the absence of the stop codon, the protein remains connected
to the tRNA. Stopping the translation reaction in ice-cold buffer with a high Mg2+ concentration stabilizes
this ternary complex, consisting of mRNA, ribosome, and nascent protein. The spacer, occupying the ribosomal
tunnel, enables the domain of interest to fold on the ribosome. These ribosomal complexes are used
for affinity selection. After washing, the mRNA of the selected complexes is released by complex dissociation.
The genetic information of binders is rescued by RT-PCR, yielding a PCR product ready to go for the
next selection cycle.
The following chemicals and enzymes are necessary
to prepare the extract and to perform ribosomedisplay
selections: Luria broth base (GibcoBRL 12795-
084); agarose (Invitrogen 30391-023); glucose (Fluka
49150); potassium dihydrogen phosphate (KH2
Fluka 60230); dipotassium hydrogen phosphate
O, Merck 1.05099.1000); yeast extract
(GibcoBRL 30393-037); thiamine (Sigma T-4625);
Tris (Serva 37190); magnesium acetate (MgAc, Sigma
M-0631); potassium acetate (KAc, Fluka 60034); L
-glutamic acid monopotassium salt monohydrate
(KGlu, Fluka 49601); 20 natural amino acids (Sigma
LAA-21 kit); adenosinetriphosphate (ATP, Roche Diagnostics
519 987); phosphoenolpyruvate trisodium salt
(PEP, Fluka 79435); pyruvate kinase (Fluka 83328); GTP (Sigma G-8877); cAMP (Sigma A-6885);
acetylphosphate (Sigma A-0262); Escherichia coli
(Sigma R-4251); folinic acid (Sigma 47612); PEG 8000
(Fluka 81268); 1,4-dithiothreitol (DTT, Promega
V3155); sodium chloride (NaCl, Fluka 71376); Tween-
20 (Sigma P-7949); neutravidin (Pierce 31000); bovine
serum albumin (BSA, Fluka 05476), Sacchoromyces
cerevisiae RNA (Fluka 83847); ribonuclease inhibitor
RNasin (Promega N211B); reverse transcriptase
Stratascript (50U/µl, Stratagene 600085-51); 10× Stratascript buffer (Stratagene 600085-52); dNTPs
(5 mM of each dNTP, Eurogentec NU-0010-50); DNA
polymerase for PCR (e.g., Vent polymerase, NEB
M0254L); PCR buffer (e.g., thermopol buffer, delivered
with Vent polymerase), dimethyl sulfoxide (DMSO,
Fluka 41640); NTPs (50mM, Sigma); nitrocefin (Calbiochem
484400); HEPES (Sigma H-3375); spermidine
(Sigma S-2501); T7 RNA polymerase (NEB M0251L);
lithium chloride (LiCl, Fluka 62476); 100% ethanol
(EtOH); sodium acetate (NaAc, Fluka 71180); heparin (Fluka 51550); disodium ethylenediaminetetraacetate
(EDTA, Fluka 03680); T4 DNA ligase (MBI Fermentas
EL0011); 4-morpholinopropanesulfonic acid (MOPS,
Fluka 69949); boric acid (Fluka 15660); guanidine
thiocyanate (Fluka 50990); N,N
(Sigma Aldrich 27.054-7); 37% formaldehyde (Fluka
47629); UHP water; if proteins are displayed that
depend on the correct formation of disulfide bonds,
protein disulfide isomerase should be used (PDI;
Sigma P3818); [35
NEG009H); triethylamine (Sigma-Aldrich 90335);
OptiPhase2 scintillation liquid (PerkinElmer
B. Bacterial Strain and Plasmid
We use E. coli
strain MRE600 for the preparation of
the extract. This strain is RNase I deficient (Kushner,
2002) and does not contain any antibiotic resistance
(Wade and Robinson, 1966).
Ribosome-display vector (pRDV), containing β-
lactamase as insert (gene bank accession: AY327136).
C. Laboratory Equipment and Hardware
The following material is used in ribosome display:
ART filter pipette tips (10 µl, 20 µl, 200 µl, 1000 µl,
nucleic acid and nuclease-free tips, Molecular Bioproducts);
QIAquick PCR purification and gel extraction
kit (QIAgen 28104 and 28704); Maxisorp plate
(Nunc-Immuno plate, Nunc 430341); step pipette
(Eppendorf Multipipette Plus 4981 000.019) with 5-
and 10-ml tips (Eppendorf 0030 069.250 and 0030
069.269); plastic seal (Corning Inc., Costar® 6524);
RNase-free 1.5-ml reaction vials (MolecularBioProducts
3445); Roche high pure RNA isolation kit (Roche
1 828 655); 0.2-mm syringe filter (Millipore
SLGPR25KS); dialysis tubing with a molecular weight
cutoff of 6000-8000Da (e.g., Spectrum Laboratories
SpectraPor 132 650).
Furthermore, standard laboratory equipment is
needed, such as Sorvall RC-5C Plus centrifuge with
rotors SS-34 and GS-3 or equivalent; refrigerated table
centrifuge; shaker incubator; 5-liter and 100-ml baffled
shake flasks for E. coli
culture; Emulsiflex (Avestin,
Canada) or French Press (American Instrument
Company, AMINCO); 4°C room; liquid nitrogen (N2
ELISA plate shaker; UV/VIS spectrophotometer;
agarose gel electrophoresis system; latex gloves,
Speed-Vac (Savant Speed Vac Concentrator SVC100H);
-20 and -80°C freezer; Scintillation counter.
Preparation of S30 Extract
General considerations: 1 liter E. coli
approximately 8 ml extract, if you plan to do ribosome
display at a large scale, grow several cultures in parallel.
It is important that the cells used for extract
preparation are harvested in an early logarithmic
phase. If the libraries used for selection contain disulfide
bonds, one should omit DTT from the extract. If
no disulfides need to be formed, 1mM
DTT can be
added to the S30 buffer as it increases the translation
Preparation of the S30 extract is performed according
to Lesley, Zubay, and Pratt, with minor modifications
(Chen and Zubay, 1983; Lesley, 1995; Pratt, 1984;
The following buffers are used in standard ribosome-
display selection rounds and we advise preparing
stocks: Tris-buffered saline (TBS; 50mM
pH 7.4, at 4°C; 150mM
NaCl), TBS with Tween [TBST;
TBS with 0.05% (500µl/l) Tween-20], washing buffer
with Tween (WBT; 50mM
Tris-acetate, pH 7.5, at 4°C; 150mM
MgAc; 0.05% Tween-20) and
elution buffer (EB; 50mM
Tris-acetate, pH 7.5, at 4°C; 150mM
NaCl; 25 mM
EDTA); 10× MOPS (0.2M MOPS,
pH 7, 50mM
sodium acetate, 10mM
EDTA); 10× TBE
buffer (89mM Tris-buffered saline, 89mM boric acid,
: The washing buffer used for ribosome display
can be adjusted to any particular requirements given
by the target molecule. Different buffer salts and detergents
are compatible, only the Mg2+
should be held at around 50 mM
. If a new buffer composition
is applied, a ribosome-display test selection
round with a known binder is recommended to determine
αtssrA: (200 µM
, 5'-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA-3', standard quality)
T7B: Forward RD primer. Introduces T7 promotor and
part of the 5' loop
SDplus: Forward RD primer. Introduces the Shine-
Dalgarno sequence and connects the T7 promoter
with the FLAG tag: (100µM
tolAk: Reverse primer for RD used with tolA as spacer
introducing a stabilizing 3' loop
Primers (reverse and forward) specific for the
library of interest, which must introduce appropriate
restriction sites for ligation into the ribosome-display
Solutions, Strain, and Hardware
strain MRE600 (Wade and Robinson,
1966); Luria broth base; incomplete rich medium:
5.6 g/liter KH2
, 37.8 g/liter K2
yeast extract, 15 mg/liter thiaminemafter autoclaving,
add 50ml 40% (w/v) glucose sterile filtered; 0.1M
MgAc; 10× S30 buffer: 100mM
Tris-acetate, pH 7.5, at
KAcmstore at 4°C or
chill buffer in ice bath before use; 10ml preincubation
mixmmust be prepared immediately before use:
Tris-acetate, pH 7.5 (at 4°C), 71µl 3M
MgAc, 75µl amino acid mix (10mM of each of the 20
natural amino acids), 0.3 ml 0.2M
ATP, 0.2 g PEP, 50 U
Material: 5-liter baffled flasks; shaker at 37°C for E.
culture; refrigerated centrifuges (GS-3, SS-34); dialysis
tubing MW cutoff 6000-8000Da; emulsiflex or
- Prepare an LB/glucose plate and streak out
MRE600 on the plate. Grow it overnight at 37°C
- Prepare all chemicals, media, and buffers for E.
coli extract preparation: Autoclave 1 liter incomplete
rich medium, 500 ml of LB/glucose medium, and one
100-ml and one 5-liter shake flask. Prepare 50ml 40%
glucose, 10ml 0.1M MgAc, and 1 liter 10× S30 buffer
(use it as 1× S30 buffer afterwards). All buffers should
be stored at 4°C.
- Prepare an overnight preculture by inoculating
50ml LB/glucose medium with a colony of MRE600,
which is shaken overnight at 37°C.
- Add 1 liter incomplete rich medium into the 5-
liter shake flask and add 50ml 40% glucose and 10ml
0.1M MgAc both by sterile filtration (0.2-µm syringe
- Inoculate the culture with 10ml overnight
culture (approximately 1%) and let it shake at 37°C to
OD600 = 1.0-1.2. Then transfer the culture in an icewater
bath and quickly add 100 g ice (small shovel) to
the culture. Shake the culture in the ice-water bath by
hand for 5 min. Collect the cell pellet by centrifugation
at 4°C (15 min, GS-3, 5000rpm). Wash the pellet at least
three times with 50-100ml of S30 buffer.
- Determine the weight of the pellets (typically
1-1.5 g/liter). The cell pellet can now be shock-frozen
in liquid nitrogen and stored at -80°C until further
processing. Do not store the pellets longer than 2 days.
Do wear gloves during all following steps!
- Thaw the pellets on ice and resuspend the cells
in S30 buffer (50ml). Centrifuge at 4°C, full speed, in
an appropriate centrifuge to collect the cell pellet. Resuspend the cells in 4ml/g (wet cell weight) S30
- Lyse the cells with one passage through an EmulsiFlex
(approximately 17,000psi) or a French press (at
1000psi). Repeating passages will decrease translation
activity. Centrifuge the lysate at 4°C for 30min (SS-34,
20,000g). Take the supernatant and repeat this centrifugation
- Transfer the supernatant to a 50-ml Falcon tube.
Add preincubation mix (1 ml/6.5ml supernatant) and
incubate at room temperature for 60min by slowly
shaking the tube. In this step most endogenous RNA
and DNA will be degraded by nucleases and translatio
n will run out.
- Transfer the extract into a dialysis device (MW
cutoff 6000-8000Da) and dialyze the extract three
times for at least 4h each at 4°C against S30 buffer
B. Premix Preparation and Extract
- Transfer the extract into a single 50-ml tube.
Aliquot the extract into RNase-free tubes immediately
(e.g., 110- and 55-µl aliquots, use filter tips to avoid
RNase contamination) and directly freeze the aliquots
in liquid nitrogen. Store the aliquoted extract at -80°C (it will be fully active for months to years).
The premix provides the S30 extract with all amino
acids (except for methionine, see later), tRNAs, the
energy regeneration system, and salts, which are
needed for translation. For optimal translation efficiency,
every premix should be adjusted to fit the corresponding
extract, especially with respect to the
concentration of magnesium, potassium, PEG-8000,
and amount of extract. The premix A recipe given later
contains only minimal concentrations of KGlu, MgAc,
and PEG-8000. By performing translations (compare
Section III,F) using a test mRNA and by gradually
adding increasing amounts of these components, the
translation efficiency of the extract will be optimized
to its maximal activity. The optimization of the premix
to the $30 extract is optimally done by translating the
mRNA encoding an enzyme, whose activity is determined
easily, such as β-lactamase. We routinely use a
cysteine-free version of the enzyme (Laminet and
Plfickthun, 1989) in a ribosome-display suitable format
(described in Section III,C) for optimization.
You will need approximately equivalent amounts of
premix and extract. Premix A: 250 mM
Tris-acetate, pH 7.5, at 4°C, 1.75 mM
of each amino acid except methionine,
ATP, 2.5 mM
GTP, 5 mM
cAMP, 150 mM
acetylphosphate, 2.5mg/ml E. coli
folinic acid, and 4 µM
α-ssrA DNA. KGlu (180-220 mM
MgAc (5-15mM), and PEG-8000 [0-15% (w/v)]
have to be adjusted to the corresponding extract. β-
Lactamase assay buffer: Dissolve 5.3 mg nitrocefin in
250µl DMSO and add this to 50ml 50mM potassium
phosphate buffer (pH 7) (Laminet and Pliickthun, 1989).
To avoid RNase contamination, use filter tips and
wear gloves for all of the following steps.
C. Preparation of the Ribosome-Display
- Mix all components to yield premix A.
- Incubate the premix in a water bath at 37°C to
solubilize all components.
- Perform different in vitro translations in parallel
of β-lactamase mRNA as described in (Section III,F).
Add increasing concentrations of MgAc (5-15mM),
KGlu (180-220mM), PEG-8000 [0-15% (w/v) of
premix], and amount of extract (30-50µl for a 110-µl
- First optimize the MgAc concentration, then the
KGlu concentration, and finally the PEG-8000 concentration.
The translation time relevant for optimization
should be around 10min. Stop translation by diluting
the reaction five times in WBT.
- To detect β-lactamase activity, use a nitrocefin
assay. Use 10-20µl of the stopped translation per milliliter
β-lactamase assay buffer and follow the reaction
with a photospectrometer at 486 nm.
- After determining the conditions giving the
highest activity, add the chemicals at the optimal concentration
to the premix A stock yielding premix Z,
optimized for this very batch of extract.
- Aliquot the premix Z in RNase-free tubes and
shock freeze the samples in liquid nitrogen (e.g.,
To perform ribosome display, one needs a highquality
library in the appropriate format. This section
does not explain how to generate this library, as this
depends entirely on the experimental goal, but rather
how to convert an existing one into a format suitable
for ribosome display.
A ribosome-display construct is composed of a T7
promoter, followed by a ribosomal-binding site and an
open reading frame, which in turn consists of the
library fused in frame to a C-terminal spacer polypeptide
that has no stop codon (Figs. 2 and 3). The lack of a stop codon prevents the binding of the termination
factors TF-1, TF-2, and TF-3. A high magnesium
concentration "sinters" the ribosome, which consists
largely of folded RNA with a protein coat. The low
temperature presumably prevents the hydrolysis of
the peptidyl-tRNA and minimizes mRNA degradation.
All these measures together ensure that the
ternary complex of mRNA (genotype), ribosome, and
displayed protein (phenotype) remains stable. The Cterminal
spacer (usually derived from tonB, tolA, M13
gpIII or pD), which will partially remain in the ribosomal
tunnel, ensures that the library protein can fold
and is displayed on the ribosome. The T7 promoter
allows efficient in vitro
transcription of the construct.
A 5' and a 3' stem loop protect the mRNA against
Solutions, Plasmids and Strains
QIAquick PCR purification and gel extraction kit;
pRDV; appropriate restriction enzymes; T4 DNA
1. Generation of the Ribosome-Display Construct
To accelerate the procedure of bringing a library
into the ribosome-display format, we generated a
vector containing the necessary flanking regions
(ribosome-display vector, pRDV; Fig. 2). The library is
PCR amplified, cut with the appropriate restriction
enzymes, and ligated into the vector such that it is in
frame with the spacer (Fig. 3). A second PCR on this
ligation product directly amplifies the library with all
features necessary for ribosome display: the T7 promoter,
the RBS, and the spacer without stop codon
(Fig. 2). This PCR product is used directly for in vitro
transcription to yield the library mRNA ready to go.
The main advantages of the ribosome-display vector
are that it can be generated in large amounts (mini to
maxi prep), it is easy to handle, and always provides
error-free library flanking regions. The use of the
vector is not only interesting for the initial generation
of the ribosome-display construct, but also for the first
selection rounds. If one only amplifies the library gene
of the selected clones after the panning procedure
without all the flanking regions, one is able to even
recover library members partly degraded by RNases
in the flanking regions. The recovered genes are then
religated into pRDV and one is again ready to go for
another round of selection.
|FIGURE 2 Generation of the ribosome-display construct. For ribosome display the library of interest has
to be flanked by an upstream promoter region and a C-terminal spacer carrying no stop codon (Fig. 3). (A)
The library is PCR amplified with primers carrying restriction sites suitable for ligation into the ribosomedisplay
vector (pRDV), which carries the necessary library flanking regions. The PCR product of the library
is digested and ligated into pRDV. A second PCR on this ligation reaction with the primers T7B and tolAk
yields a PCR product ready for in vitro transcription. (B) Alternatively, the ribosome-display construct can be
generated by assembly PCR. The library and the spacer are PCR amplified separately with primers so that
the C-terminal part of the library and the N-terminal part of the spacer share overlapping sequences. An
assembly PCR with the library and the spacer DNA, using appropriate primers, finally yields the ribosomedisplay
construct. In vitro transcription of the PCR product of either A or B yields mRNA carrying 5' and 3'
stem loops (which make the mRNA more stable toward exonuclease digestion), a ribosome-binding site
(RBS), the library of interest, and a spacer carrying no stop codon. By stopping the in vitro translation in icecold
buffer with high Mg2+ concentration, stable ternary complexes of mRNA, ribosome, and nascent protein
are formed, ready for panning.
|FIGURE 3 DNA sequence of the expression cassette of the ribosome-display vector (pRDV). mRNA is
produced from a T7 promoter starting with a 5' stem loop, with no additional overhang. The ribosomebinding
site (RBS), also called Shine-Dalgarno sequence, is located upstream of the start codon. The open
reading frame consists of a FLAG tag, the β-lactamase gene (serving as a dummy insert) in frame with a
protein spacer, here tolA. Different restriction sites allow cloning of the library into pRDV to replace the
2. Generation of the Ribosome-Display Construct
via Assembly PCR
- Amplify the library with primers carrying the
appropriate restriction sites for cloning into pRDV
- Digest the PCR product and purify it using
QIAquick columns (≥150 ng; amount depending on the
library size), pRDV is digested, optionally dephosphorylated,
and the backbone is agarose gel purified.
The vector insert has to be removed, as it would also
ligate to the library DNA, decreasing the final
- Ligate the PCR product of the library into pRDV
(molar ratio insert: vector = 7:1).
- Perform a PCR reaction on the ligation mix with
the forward primer T7B (annealing on the T7 promoter
with a stabilizing 5' loop) and the reverse primer tolAk
(annealing on the C-terminal spacer carrying a stabilizing
- Analyze the DNA on an agarose gel, checking
size, purity, and amount. If the band is sharp and indicates
a concentration higher than 40ng/µl, the PCR
product is used directly (i.e., without purification) for in vitro transcription.
In some cases, it may be preferable not to ligate the
library into pRDV, but to use PCR assembly to generate
the ribosome display construct (Fig. 2). In this case,
both the library and the spacer (e.g., tolA) are PCR
amplified so that the 3' end of the library and the 5'
end of the spacer share overlapping sequences (Fig. 2).
The PCR products of the library and the spacer are
assembled and amplified with the primers SDplus
(introducing the ribosome-binding site and the connection
to the T7 promoter) and tolAk. A final PCR
reaction with the primers T7B (introducing the T7 promoter)
and tolAk completes the construct, ready for
transcription (Fig. 2).
D. Transcription of PCR Products
Solutions and Hardware
- Amplify the library of interest with appropriate
primers, introducing the FLAG tag at the 5' end of the
library, on which the primer SDplus can anneal in
step 3. The reverse primer should anneal on the library
and add an overlap corresponding to RDlinktolA.
There is no need to introduce restriction sites; however,
we strongly recommend the use of the same primers
as for the cloning into the RDV to have the possibility
of using pRDV and to reduce the number of different
- Amplify the ribosome-display spacer using the
primers RDlinktolA (forward) and tolAk (reverse).
Thereby, the forward primer generates an overlap
between the library and the spacer.
- In an assembly PCR reaction, the spacer is fused
to the library. Mix DNA of the library with an excess
of spacer DNA and perform 7 cycles of PCR without
the addition of primers. For this step, we use a lower
annealing temperature as for the normal amplification.
After 7 cycles, add primers tolAk and SDplus to
the reaction and perform another 25 cycles of
- Isolate the full-length band from an agarose gel
and amplify the band using the primers T7B and tolAk.
- Analyze the DNA on an agarose gel, checking size, purity, and amount. If the band is sharp and indicates
a concentration higher than 40ng/µl, the PCR
product is used directly (i.e., without purification) for in vitro transcription.
5× T7 polymerase buffer: 1M
HEPES-KOH, pH 7.6,
MgAc, 10 mM
spermidine, 200 mM
each); T7 RNA polymerase; RNasin; 6M
70% EtOH; 100% EtOH; 3M
NaAc; agarose; guanidine
thiocyanate; formamide; 37% formaldehyde; MOPS;
Speed-Vac; heating blocks; UV/VIS spectrophotometer.
To avoid RNase contamination, use filter tips and
wear gloves for all of the following steps.
E. Target Molecule Immobilization
- Mix the components for the following reaction
using the PCR product from Section III,C. The PCR
product should not be purified. Add 20.0 µl 5× T7 polymerase
buffer, 14.0µl NTPs (50mM each), 4.0µl T7
RNA polymerase, 2.0µl RNasin, 22.5µl PCR product
(unpurified PCR reaction), and 37.5 µl UHP water. Let
the transcription reaction run for 2-3 h at 37-38°C.
- Add 100 µl UHP water and 200 µl 6M LiCl, both
ice cold, and place on ice for 30 min, before centrifugation
at 20,000g (4°C, 30min). Discard supernatant and
wash the pellet with 500 µl ice-cold 70% EtOH. Dry the
pellet on the bench (5 min, open lid) and take it up in
200µl ice-cold UHP water. Make sure it is dissolved
completely before centrifuging at 20,000g (4°C, 5 min).
- Transfer 180µl supernatant to a new tube and
add 20µl 3M sodium acetate and 500µl ice-cold 100%
EtOH. Keep the solution on ice or at -20°C for 30min
before centrifuging it at 20,000g (4°C, 30min). Discard
the supernatant and wash the pellet with 500µl icecold
70% EtOH. Discard the supernatant and dry the
pellet in a Speed-Vac apparatus.
- Take up the mRNA pellet in 30µl ice-cold UHP
water and make sure it is dissolved completely. Take
2µl of this solution and dilute to 500µl with ice-cold
UHP water for OD260 quantification and immediately
N2 freeze the rest of the RNA for further use.
- Immediately (the RNA will be degraded and the
signal will increase) measure the OD260. For RNA, an
OD260 of 1 corresponds to a concentration of 40µg/ml.
- Add UHP water to the RNA stock in order to
reach a standard concentration of 2.5µg/µl.
- The RNA quality can optionally be checked by
agarose gel electrophoresis. Cast an agarose gel (1.5%,
depending on your RNA size) adding 2% (w/v) of
1M guanidinium thiocyanate. Denature 5 µg (i.e., 2 µl) RNA for 10 min at 70°C in 15.5 µl sample buffer (10 µl
formamide, 3.5µl 37% formaldehyde, 2µl 5× MOPS),
place it on ice, add 2 µl DNA loading buffer (50% glycerol,
1 mM EDTA in H2O), and run the gel.
To perform a selection, the target molecule must be
immobilized in a conformation relevant for further
applications. Typically, a protein will have to be in its
native conformation. A very promising way to achieve
this to biotinylate the target molecule and immobilize it
via neutravidin or streptavidin. Biotinylation can be
done chemically with commercially available reagents,
either attaching the biotin to cysteine or lysine residues.
The problem of this unspecific approach is that biotinylation
might destroy epitopes. Alternatively, the target
protein can be expressed in recombinant form with a
biotinylation tag, i.e., a peptide sequence, which is recognized
and biotinylated by the E. coli
enzyme BirA (Schatz, 1993). If for any reason biotinylation
is not an option, one can either immobilize the
target molecule directly on the hydrophobic surface of a
microtiter plate well or use a specific antibody, which
itself can be immobilized easily via protein A or G. Note
that the buffers used here may have to be adapted to the
needs of the target molecules.
Solutions and Hardware
TBS, TBST, WBT at 4°C
Maxisorp plate; plastic seal; step pipette; plate shaker;
stock neutravidin; BSA; target molecule of
To avoid RNase contamination, use filter tips and
wear gloves for all of the following steps. We also recommend
carrying out the selection, RT and PCR in
duplicate, to check the reproducibility of the selection.
Therefore, one target molecule is routinely immobilized
in two wells.
Day 2 (Day of the Ribosome-Display Round)
- Wash a Maxisorp plate three times with TBS and
beat dry. Pipette (with a step pipette) 100µl of a 66 nM neutravidin solution in TBS into the wells. Seal with
plastic and store overnight at 4°C. If the target molecule
is not biotinylated it can also be directly immobilized
by the same procedure as neutravidin, but it
might denature at least partly during this procedure.
F. In Vitro Translation
- Wash the plate incubated overnight with cold
TBS three times. Add 300µl 0.5% BSA in TBS to the
wells (with a step pipette) to block all hydrophobic surfaces with BSA. Seal the plate with plastic and incubate
on a shaker for 1 h at room temperature. Alternatively,
sensitive or fragile targets should be incubated
for 2.5h at 4°C. If the target molecule is immobilized
directly, incubate the BSA solution for 2h at 4°C and
proceed directly to step 5.
- Wash the plate three times with TBS, beat dry,
and add 200µl 0.5% BSA in TBS to each well (step
- Add 5µl of the target molecule (~10µM) to the
respective well. Use a biotinylated target molecule
void of free biotin. Seal with plastic and incubate on a
shaker for 1 h at 4°C. To avoid binders to BSA or neutravidin,
it is recommended to immobilize the target
molecule only in every second well and use the alternate
wells for prepanning.
- Wash the plate four times with TBS to get rid of
the unbound target molecule. Wash at least once with
WBT (step pipette) to equilibrate the well with the
ribosome-display buffer. Incubate wells that will not be
used directly for panning with WBT (amount of liquid
as will be used for panning). Keep the plate at 4°C.
UHP water; WBT; WBT with 0.5% BSA; heparin (200
mg/ml); methionine (200mM
); S30 extract; premix
Z; the mRNA of the library; optionally PDI (4
mg/ml, reconstituted from lyophilized protein); all
buffers should be kept on ice, the RNA in liquid
nitrogen unless stated otherwise
We recommend carrying out the panning in duplicate
to check the reproducibility of the selection. To
avoid RNase contamination, use filter tips and wear
gloves for all of the following steps.
Solutions and Hardware
- Mix the following components (amounts given
are for one reaction): 13.0µl UHP water, 2.0µl Met
(200mM), 41.0µl premix Z (thaw on ice, vortex before
pipetting), and 50.0µl extract. Volumes might vary,
depending on the batch of S30 extract. This mix can be
kept on ice for a short period (few minutes). If disulfide
bridges are to be formed, add 0.625 lal PDI (4 mg/ml).
- Add 4µl mRNA (2.5µg/µl) into a fresh RNasefree
tube and freeze it in liquid nitrogen.
- Add the translation mix to the frozen mRNA,
dissolve the pellet by flicking the tube, and translate
for 6-12min at 37°C.
- In the meantime, prepare 440 µl stopping buffer
[WBT with 0.5% BSA and 12.5 µl/ml heparin (200mg/
ml)] in RNase-free tubes and put them on ice.
- After the 6- to 12-min translation, pipette 100µl
translation into the ice-cold stopping buffer to stop
translation (always keep the ribosomal complexes on
ice or at 4°C.
- Centrifuge at 20,000g, 4°C for 5 min and use the
supernatant containing the ribosomal complexes for
WBT; EB; yeast RNA (25µg/µl); cold room; microtiter
plate shaker; Roche High Pure RNA isolation kit
All the following steps should be performed in a
cold room (at 4°C or slightly below). The low temperature
guarantees complex and mRNA stability. Under
such conditions ribosomal complexes have survived
off-rate selection procedures of 10 days and longer.
During all binding and elution steps, shake the
microtiter plate gently. To avoid RNase contamination,
use filter tips and wear gloves.
Solutions and Hardware
- Add 100-200µl stopped translation mix to the
well, where no target molecule is immobilized and
incubate for 1 h. In this prepanning step, all BSAbinding,
neutravidin-binding, or simply sticky complexes
are removed. If unspecific complexes are
causing problems, more than one prepanning step
should be done.
- Transfer the solution to the well with the immobilized
target molecule and incubate for 1 h.
- Wash the well to remove nonbinding complexes.
The time span of the washing step and the number of
washing steps define the selection pressure. One
usually starts with short washing times in the first
rounds (only rinsing six times) and increases the
washing periods for later rounds (up to 3-4h). As
washing is always a dilution, it is important to fill the
wells to the top and exchange the buffer many times
(at least six times, in all rounds!). If harsher selection
pressure than simple washing is required, e.g., to select
for affinities below nanomolar, one should consider
immobilisation of low target molecule concentrations,
competitive elution (with free target molecule in the
solution), or off-rate selection procedures (see Section
- Before the mRNA of the selected complexes is
eluted, prepare three RNase-free tubes for each well to
be eluted: one tube with 400µl lysis buffer of the
Roche RNA purification kit to purify the RNA, one
tube to collect the purified mRNA, and one tube for
reverse transcription. Also prepare one Roche-RNA purification column for each well to be eluted. Label
all tubes and columns appropriately.
- Calculate and prepare the amount of elution
buffer you need (200 µl/well) and add 50µg/ml S. cerevisiae
RNA (2µl/ml of a 25-µg/µl stock) to it. Keep the
buffer on ice.
- Elution is done in the cold room. Wash your well
one last time with WBT, remove the supernatant completely,
and beat the plate dry. Add 100µl elution
buffer, shake for 10 min, transfer this eluate to the tube
containing the lysis buffer, and mix well. Repeat this
procedure with another 100µl of elution buffer. In the
lysis buffer, the RNA is stable and can be brought to
Roche High Pure RNA isolation kit; 100mM
RNasin; Stratascript (50U/µl; Stratagene); dNTPs;
DNA polymerase; oligonucleotides; 10× Stratascript
buffer; polymerase buffer; DMSO; agarose
The RNA purification is done according to the
Roche protocol, with slight modifications. All the centrifugation
steps are carried out at 4°C.
- Just before RNA purification, thaw the reagents
for reverse transcription (DTT, 10x Superscript buffer,
dNTPs, oligonucleotide for reverse transcription).
- Set two heating blocks to 70°C and 50°C, respectively.
- Apply the lysis buffer/eluate mixture on the
column and spin for I min at 8000 g. Discard the flowthrough
and wash with 500µl buffer 1 (black-capped
bottle in the Roche kit; the DNase incubation step,
described in the Roche protocol is not necessary).
Discard the flow through and wash with 500 µl buffer 2
(blue-capped bottle in the Roche kit). Discard the flow
through. Add 100µl buffer 2 (blue cap) and spin for
2min at 13000g. Transfer the column to a tube to collect
- Elute with 30µl Roche elution buffer (at 8000g for
1 min) and directly put the RNA containing collection
tube to 70°C for 10min to denature the RNA. During
this incubation time, pipette the reverse transcription
(i.e., directly proceed with step 5).
- In a master mix tube, add the following components
(amounts given are for one reaction): 0.25µl reverse primer (100µM), 0.5µl dNTP (5mM each),
0.5 µl RNasin, 0.5 µl Stratascript, 2.0 µl 10× Stratascript
buffer, 2.0 µl DTT (100 mM), and 2.0 µl UHP water. Distribute
the RT mix (7.75µl) to the previously labelled
tubes and keep them on ice.
- Spin the denatured eluted RNA samples shortly
and set them on ice. Add 12.25 µl of the eluted RNA to
the RT-mix (N2 freeze the rest of the RNA directly after
adding it to the reaction).
- Place the RT reaction on a 50°C heat block for
- Mix the following components (amounts given
are for one reaction): 5.0µl Thermopol buffer, 2.0µl
dNTPs, 2.5µl DMSO, 1.0µl forward primer (100µM),
1.0µl reverse primer (100µM), 5.0µl RT template,
0.5 µl polymerase, and 33.0µl UHP water. PCR results
can be improved if a hot start is performed (adding the
polymerase in the 5-min preincubation step at 95°C).
- Run the following PCR program (to be adapted
according to primers and template): 5 min at 95°C, X
times (30s at 95°C, 30s at 50°C, 1 min at 72°C), 5 min
at 72°C, and 4°C infinitely. Adjust the number of cycles
(X = 25-45) to the corresponding selection round.
- Verify the PCR product quality by an appropriate
agarose gel electrophoresis.
- Usually the quality of the PCR product is not
good enough to be used directly for in vitro transcription
to generate mRNA for the next round. Gel purify
the desired band and perform a second PCR on the
purified first PCR to yield high-quality DNA.
- If you wish to check the pool of selected clones
for binders, ligate the selected library members after
PCR amplification into a vector suitable for expression,
transform E. coli to obtain individual clones, and test
binding specificity, e.g., by crude extract ELISA. To
assess the specificity of the binders, one can either
compare binding of the selected molecules to the specific
target molecule with the binding to an unrelated
molecule or test that the specific binding can be inhibited
by adding the purified unbiotinylated target molecule.
The level of inhibition gives a first crude
estimate of the affinity.
. If the RT-PCR with the forward (e.g.,
T7B) and reverse (e.g., tolAk) primers does not yield a
high-quality product, one can amplify only the coding
region of the selected library members. This usually
improves the yield and the quality of the PCR product.
This is most likely due to the fact that RNases degrade
the mRNA from the ends. Amplifying only the central
(library) stretch can rescue partly degraded clones. The PCR product of the library stretch is subsequently religated
into pRDV as described in Section III,C. As this
procedure rescues more clones than the PCR of the
whole construct, we would recommend it for the first
rounds to guarantee that no binders are lost.
. Ribosome display has many
error-sensitive steps. It is therefore recommended to
do panning, RT, and PCR in duplicate to check the
reproducibility of the selection. Specific binders should
be enriched from round to round. This should correlate
with the number of PCR cycles needed to amplify
the DNA after RT, which should decrease from round
to round. Usually, one can reduce the cycle number by
around five per round. If the selection pressure is
increased, the yield will drop. When enrichment is
observed (for designed ankyrin repeat protein
libraries, (Binz et al.
, 2004) e.g., after round two, while
for antibody libraries after round three to four), one
can test the specificity of the selected pool by comparing
panning results against the correct and an unrelated
target molecule. If the pool is specific, only the
correct target molecule will give a PCR product after
RT. If the pool also gives a signal with the unspecific
target molecule, the majority of the clones are still
unspecific. However, there may also be a population
of specific binders in the pool. An additional panning
round with increased prepanning can reduce the background.
Alternatively, single clone analysis might
directly yield specific binders.
I. Radioimmunoassay (RIA)
Radioimmunoassay is another fast and convenient
method to check whether specific target-binding molecules
have been enriched in a pool (Hanes et al.
It can be used for the evaluation of both selected pools
and individual binders. RNA of a pool or single binder
is translated in the presence of radioactively labelled
[35S]-methionine. Therefore, the radioactive protein
that binds to the surface-immobilized target molecule
can be quantified easily. The binding should be performed
in the presence and absence of soluble competitor
target molecules, and a control for unspecific
binding should be included. In the competition assay,
the minimal concentration of competitor still leading
to half-inhibition of the maximal binding signal is a
crude measure for the affinity. Therefore, RIA facilitates
the ranking of the affinities of different clones
isolated after affinity maturation.
Solutions and Hardware
The general handling is the same as for the in vitro
translation. However, the radioactive material must be handled with the appropriate precautions. Do the
radioactive work in a designated area of your laboratory.
We recommend using filter tips and wearing
gloves during the experiment. We recommend doing
the RIA in duplicate.
UHP water; WBT; WBT with 0.5% BSA; milk powder;
heparin (200mg/ml); [35
1175Ci/mmol); S30 extract; premix Z; mRNA of a
selected pool or single binders; pRDV; optionally
PDI (4mg/ml); 0.1M
triethylamine; liquid scintillation
Scintillation counter, ELISA plate shaker
- The analysis of whole pools of potential binders
can be performed similarly to a normal ribosomedisplay
round. A PCR reaction is performed on the
selected pool but with primers introducing a 3' stop
codon and the standard ribosome display 5' end
(including T7 promotor and ribosome-binding site).
From this PCR product, RNA is produced as described
in Section III,D.
- Alternatively, if you wish to analyze single
binders, digest the PCR product from step 1a with the
appropriate restriction enzymes and ligate into the
ribosome-display vector (RDV). After transformation,
isolate plasmids from single colonies. The transcription
can be performed directly from the plasmid,
which should be present at a concentration of at least
- Prepare a Maxisorp plate with immobilized
target molecules as described in Section III,E.
- Perform in vitro translations as described in
Section III,F with the following modifications: instead
of cold methionine use 2 µl of [35S]-methionine (0.3 µM,
50µCi/ml final concentration) per 110-µl reaction and
perform translation for 30-45 min at 37°C.
- Stop the translation with 440µl WBT and centrifuge
the samples for 5 min at 14,000rpm, 4°C.
- Aliquot the supernatant and dilute the samples
with 4% milk in WBT to a final concentration of 1%
milk. For inhibition studies, different concentrations of
competitor should be added to the different aliquots
and the mixture should be equilibrated for 1 h at room
temperature prior to step 6.
- Split the samples to at least duplicates and apply
them to the blocked plate. Let the protein bind to the
target molecule by shaking slowly for maximally
- Wash the plate rigorously 5-10 times with WBT.
- Elute bound protein with 100µl of a 0.1M solution
of triethylamine (10min at room temperature).
- Transfer the eluates into scintillation tubes containing
5 ml scintillation solution "OptiPhase2."
- Quantify the radioactivity in a scintillation
This section states some observations we have made
during the years of performing ribosome display and
gives a summary of how ribosome display can be used
for directed evolution experiments.
A. Selection from Naive Libraries
Naïve libraries, in our hands synthetic antibody
libraries or designed repeat protein libraries, are a difficult
challenge for selection experiments. The task is to
select the specific binders, which are few in numbers,
out of a very large number of nonbinders. The outcome
of such experiments is not only dependent on the presence
of high-affinity binders in the library, but also on
the behavior, or "stickiness," of the rest of the library
population. In other words, selection must be directed
toward specific binding, in contrast to nonspecific
binding. This can be achieved with experimental tricks,
such as introducing a prepanning step, or by trying to
reduce the stickiness of the library population. For
selection with naive scFv libraries, it has turned out
that six selection rounds were necessary to obtain specific
binders (Hanes et al.
, 2000b). The designed ankyrin
repeat protein libraries routinely yield binders after
only three to four rounds (Binz et al.
, 2004). We suspect
this might be due to the fact that these repeat proteins,
which are extremely well expressed in E. coli
, fold well,
and are very stable, are also displayed better in vitro
than scFv fragments, which are intrinsically somewhat
more aggregation prone.
B. Affinity Maturation of Binders
evolution, the alternation of diversification
and selection, is a powerful strategy used to improve
proteins. Ribosome display is an ideal platform to
perform such experiments. It allows very fast selection
cycles and the PCR step is ideal to generate diversity
in between the selection rounds. This diversification of
the selected pools by random mutations increases the
sampled sequence space. Error-prone PCR, e.g., using
concentrations (Leung et al.
, 1989), imbalanced dNTP concentrations (Cadwell and Joyce, 1994),
or nucleotide analogues (Zaccolo and Gherardi, 1999;
Zaccolo et al.
, 1996), is one strategy often applied to
achieve diversification. Another powerful strategy to
create diversity is DNA shuffling on the selected pools
in between the rounds (Stemmer, 1994).
When compared to a selection experiment from a
naïve library, the challenge in affinity maturation is different.
The applied library will usually be created from
a single clone or a pool of clones, which are already
good binders. Since in affinity maturation we do not
wish to select for binding as such but for better
binding, we need an adjustable selection pressure (Jermutus et al.
, 2001). In principle, two strategies can be
used to select tight binders out of a pool of binders.
The first affinity maturation strategy is to supply
very little of the immobilized antigen. The concept is
that the binders all compete with each other and, at
equilibrium, the tight binders keep the binding spaces
occupied. In practice, however, there are several complications
to this concept. At extremely low target
molecule concentrations, the relative proportion of
unspecific binding sites gets very high (BSA, neutravidin)
so that great care has to be taken to avoid
unspecific binders. Also, if medium-affinity binders
outnumber the tight binders, the enrichment will be
very slow. Finally, if binding is very tight, the equilibrium
is reached exceedingly slowly.
The second very successful strategy for affinity
maturation is off-rate selection. The assumption
fundamental to off-rate selection is that the on-rate of
most protein-protein interactions is in the range of
(Wodak and Janin, 2002) and proteinligand
interactions in the range of 106
that the affinity is largely governed by the off-rate. By
first incubating the ribosome-displayed polypeptide
with the biotinylated target molecule (typically for 1 h)
followed by the addition of a large excess of nonbiotinylated
target (1000-fold excess), the selection pressure
is governed by the dissociation of the binders
from the biotinylated target. While tight binders will
remain bound to the biotinylated target, others will
dissociate and then rebind to the excess of unbiotinylated
target. The incubation time equals the selection
pressure and should be adjusted to the expected offrate.
If the recovery is poor, it is often useful to include
a nonselective round to enrich the binders.
C. Evolution of Properties Other Than High-
The selection strategies for molecular properties
other than high-affinity binding were summarised in
Section I. A library can be evolved for these properties, just as it can be for affinity. It exceeds the scope of this
chapter to discuss each strategy in detail, and there are
many more possibilities that have not been explored
The authors thank all former and present members
of the Pliickthun laboratory involved in ribosome
display who helped in developing the present
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