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1
A nanobody-based strategy for rapid and
scalable purification of human protein
complexes
In the format provided by the
authors and unedited
Supplementary Table
1
.
Publicly or commercially available GFP
-
tagged plasmids, cell
lines or transgenic
organisms.
Category
Organism
Kind
Reference
Source
Fungi
S. cerevisiae
4
,
159 strains
Huh et al.,
2003
5
Thermo Fisher
http://bit.ly/3EYKoHl
S. pombe
1
,
058 strains
Hayashi et al.,
2009
6
NBRP
http://bit.ly/3SJIK24
Invert
e
brates
C. elegans
14,637
constructs
Sarov et al.,
2012
7
TransgenOme
http://bit.ly/3KYDSEi
> 100 strains
N/A
CGC
http://bit.ly/3IQYpIn
D. melanogaster
23,169
constructs
Sarov et al.,
2016
8
TransgenOme
http://bit.ly/3KYDSEi
880 lines
Sarov et al.,
2016
8
VDRC
http://bit.ly/3KTmNvo
Mammals
Mouse
ES cell lines
Poser et al.,
2008
9
58,000 ORFs
N/A
origene
http://bit.ly/3ZAEDrg
Rat
18,000 ORFs
N/A
origene
http://bit.ly/3ZAEDrg
Humans
78,000 ORFs
N/A
origene
http://bit.ly/3ZAEDrg
HeLa cell lines
Poser et al.,
2008
9
N/A
1,125 HeLa
cell lines
Hein et al.,
2015
10
N/A
Thousands of plasmids encoding GFP
-
and ALFA
-
tagged proteins from various organisms can also be obtained from
Addgene.
ORF = open
-
reading frame
. ES = embryonic stem cell.
Supplementary Table
2.
Selection of previously characterized affinity binder pairs that could be used for
TagON/OFF purifications.
Ta
g
Binder
Reference
GFP
variants
13x ‘LaG’ nanobodies that all bind to
A. victoria
GFP
and its derivatives CFP, BFP, and YFP. 11x of those
also bind to
A
.
m
acrodactyla
CFP.
Fridy et al.,
2014
13
RFP variants
6x ‘LaM’ nanobodies that all
bind to mCherry, 1x also
binds to DsRed.
C
-
tag
Nanobody that binds C
-
terminal ‘EPEA’ peptide tag,
but also binds to endogenous human
α
-
synuclein
De Genst et al.,
2010
14
Spot/BC2
-
tag
Nanobody that binds
PDRVRAVSHWSS
’ peptide,
also called BC2 tag, but also binds to endogenous
human
β
-
catenin
Braun et al.,
2016
15
;
Virant et al.,
2018
16
PepTag
PepTagNB a nanobody that binds the
α
-
helical
peptide ‘
AVERYLKDQQLLGIW
’ derived from HIV
glycoprotein 41 (gp41)
Strokappe et al.,
2019
17
; Traenkle et al.,
2020
18
NanoTag
VHH05
NbVHH05
nanobody
that
binds
‘QADQAEKELARQIS’ peptide tag
Xu et al.,
2022
19
NanoTag
127D01
Nb127D01 nanobody that binds
‘SFEDFWKGED’
peptide tag
Rabbit IgG
2x anti
-
rabbit IgG nanobodies, for purification of IgGs
or IgG targets
Pleiner et al.,
2018
52
Mouse IgG
29x anti
-
mouse IgG nanobodies, light chain and
subclass specific binders, for purification of IgGs or
IgG targets
ED or ZZ
domain dimer
from Protein A
ZpA963 affibody dimer ( ̃13.5 kDa) binds ED or ZZ
domain dimer tags with 16
pM affinity
Lindborg et al.,
2013
20
;
Vera Rodriguez et al.,
2019
4
Colicin E7
Immunity protein 7 binds DNase deficient Colicin E7
with very high affinity (K
D
̃
10
-
14
-
10
-
17
M
)
Vassylyeva et al.,
2017
21
Supplementary Table 3
.
Anti
-
GFP and anti
-
ALFA nanobodies withstand harsh buffer conditions.
Condition
GFP Nb
A
LFA
Nb
Urea
8 M
6 M
Triton X
-
100
1%
1%
SDS
1
%
0.1%
GuHCl
4
M
2 M
NaCl
2 M
3 M
DTT
1 mM
100 mM
Deoxycholate
N/A
1%
Glycerol
30%
N/A
NP
-
40
2%
N/A
TCEP
0.2 mM
N/A
MgSO
4
N/A
1 M
DDM
N/A
1%
Comparison of buffer conditions tolerated by
the anti
-
GFP
nanobody Enhancer
(
https://bit.ly/3kLbpHr
)
and
the anti
-
ALFA
ST
nanobody
3
.
Nb = nanobody; N/A = data not available.
a
b
TCTAGT
GGATCC
GGTTCT
GCTAGC
GGTTCAGGTTCT
ACCGGT
GGATCA
TCTAGA
TAA
CTCGAG
AGATCA
CCTAGG
CCAAGA
CGATCG
CCAAGTCCAAGA
TGGCCA
CCTAGT
AGATCT
ATT
GAGCTC
BamHI
G
S
S
pTS93
pTS93-102 MCS
pTS103-116 MCS
S
G
S
A
S
G
S
G
S
T
G
G
S
S
R
*
NheI
AgeI
XbaI
BamHI
NotI
G
M
S
G
S
A
S
G
S
G
S
T
G
G
S
S
R
T
S
NheI
AgeI
XbaI
XhoI
GCGGCCGC
CATG
GGATCC
GGTTCT
GCTAGC
GGTTCAGGTTCT
ACCGGT
GGATCA
TCTAGA
ACTAGC
CGCCGGCG
GTAC
CCTAGG
CCAAGA
CGATCG
CCAAGTCCAAGA
TGGCCA
CCTAGT
AGATCT
TGATCG
5’ LTR
RRE
MCS
cPPT
WPRE
Amp
R
GFP
3’ LTR
ORI
pTS103
Amp
R
MCS
3’ LTR
cPPT
WPRE
5’ LTR
RRE
CMVTetO2
CMVTetO2
GFP
ORI
Supplementary Figure 1. Sequence elements and multiple cloning site (MCS) of lentiviral transfer plas
-
mids pTS093 and pTS103.
(
a
) Schematic of pTS93, with detailed sequence view of the MCS for
pTS93-pTS102. (
b
) Schematic of pTS103, with detailed sequence view of the MCS for pTS103-116 (Kozak
sequence underlined). 5’ LTR = 5’ long terminal repeat, RRE = Rev response element, cPPT = central polypurine
tract, CMVTetO = cytomegalovirus promoter with 2x TetO elements, MCS = multiple cloning site, WPRE =
Woodchuck hepatitis virus post-transcriptional regulatory element, 3’ LTR = 3’ long terminal repeat, Amp
R
=
Beta-lactamase (ampicillin/carbenicillin resistance), ORI = origin of replication.
Supplementary Data 1: A c
loning guide for
the
pTS
0
93
-
116
lentiviral
transfer
plasmid
toolbox
The pTS
0
93
-
116 lentiviral transfer plasmid toolbox
was created to allow easy implementation of
our anti
-
GFP
and anti
-
ALFA nanobody
-
based purification strategy. It
contains plasmids that allow
fusion of a protein of interest (POI) to either N
-
or C
-
terminal GFP or ALFA peptide tags with or
without additional proteas
e cleavage sites. This guide describes how to clone your POI into these
plasmids
to create a
transfer plasmid
that
can be co
-
transfected with the packaging plasmids
psPAX2 (
Addgene #12260)
and pMD2.G (
Addgene #12259)
into Lenti
-
X
293T
cells
to create
lentivirus
.
The resulting lentiviral supernatant can then be used to transduce human Expi 293F
suspension cells
or many other cell lines
to express your tagged POI for purification trials.
All
transfer
plasmids contain the same multiple cloning site (MCS)
with
4 unique restriction sites
(BamHI, NheI, AgeI and XbaI)
, as well as
5
NotI and
3
XhoI s
ites
(
Supplementary
Fig
ure
1
)
.
Using the same
restriction site
, multiple plasmids encoding the POI fused to either GFP or ALFA
tag at N
-
or C
-
terminus can therefore
easily
be generated
d
uring initial optimization trials. Besides
restriction enzyme digest, w
e routinely clone
POIs
into these plasmids using
G
ibson
assem
bly
.
To choose
a
method, consider the following:
Restriction enzyme cloning
Gibson cloning
Template
DNA
features
Insert
sequence needs to lack the
chosen
MCS
restriction sites
Internal restriction sites in
insert don’t interfere
Modularity
Modular, digested i
nsert
fragment
can
be
ligate
d
into any plasmid
Not modular, d
ifferent primers
needed to clone
insert
into
different
backbones
Speed
Restriction
digest
adds extra step
after PCR
There’s no
need to digest
the
PCR product
Primer
length
Re
quires shorter primer
overhangs
(6 bp flanking + 6
bp restriction
site)
Requires longer primer
overhangs (15
-
40 bp Gibson
overhangs)
General p
lasmid design considerations
:
Make
sure to include a stop codon in your reverse primer when cloning into pTS
0
93
-
pTS102
.
T
here is a stop codon after the MCS but at
least 2 amino acids will be added if no stop codon
is added to the insert fragment
.
When adding an N
-
terminal tag (
using
pTS
0
93
-
p
TS102) check the
U
niprot annotation
(
https://www.uniprot.org
) of
your POI to see if the initiator methionine is removed. If so, it is
best to avoid including this codon in your insert
.
When adding a C
-
terminal tag, the N
-
terminus of your POI will include additional amino acids
from the MCS sequence as well as a
n
initiator
methionine that we have included in the
backbone to ensure a functional
K
ozak
consensus
sequence
(5’
-
(gcc)gccRcc
ATG
G
-
3’)
.
If it is important to
maintain
the
endogenous N
-
terminus, you
can
digest your backbone with
NotI
,
but you must ensure that t
he resulting sequence contains a functional
K
ozak sequence.
Restriction enzyme cloning
Choosing r
estriction
sites:
1.
Check the sequence of your
POI
DNA
template
for BamHI, NheI, AgeI, and XbaI
recognition
sites
and choose
2 sites in the MCS that
do
not
occur
in your
template
.
2.
If possible,
choose the outermost sites to
avoid adding excess amino acids to the N
-
or C
-
terminus of your POI (see
Supplementary
Fig
ure
1
)
.
3.
A
void using
both
NheI and XbaI when possible because these two enzymes
create
matching
overhangs
. This thus
reduc
es
the chan
c
e of successful ligation
in the correct orientation
.
Designing primers:
1.
Make sure
that you
are using the correct reading frame of the template DNA and that it does
not contain premature stop codons.
2.
Design both
forward and reverse primers
to
be between 20
-
40 b
ase
p
airs (bp)
in length and
to
have
a melting temperature (T
M
)
between
5
8
-
64
.
M
ake sure each primer
ends in
a
G
or
C
at the
3’ end
.
3.
The 5’ end of each primer must contain a string of 6 nucleotides (of any sequence) followed
by the restriction site. This is needed to provide a toehold for the restriction enzyme.
4.
Design
the
reverse
pr
imer
to be reverse complement of the template DNA sequence
.
5.
Check
primers for both intra
-
and intermolecular
high T
M
hairpins using
e.g.
IDT
’s
oligoanalyzer (
https://www.idtdna.com/pages/tools/oligoanalyzer
)
and introduce changes to
remove these if necessary
.
Run PCR reaction:
1.
Mix reaction components in a 0.2 ml PC
R tube on ice
PCR reaction
(50
μl)
Volume (μl)
Reagent
25
Q5
High
-
Fidelity
2x
M
aster
M
ix (NEB, USA)
1
10 ng/μl template
0
.5
5
0 μM forward primer
0
.5
5
0 μM reverse primer
23
ddH
2
O
2.
Program
thermocycler with the
following
settings and run PCR
:
Thermocycler settings
#
Step
Temperature
Time
1
Initial denaturation
98°C
30 sec
2
3
4
Denaturation
Annealing
Extension
98°C
58
-
64°C
72°C
10 sec
30 sec
30 sec per 1 kbp
30x
cycles of
steps 2
-
4
5
Hold
4
-
10°C
kbp = kilo base pair
3.
Purify reactions using Q
IAquick
PCR
Purification
k
it (QIAGEN, Netherlands)
4.
Elute in 42 μl ddH
2
O
Double restriction enzyme digest of purified PCR product:
1.
Set up restriction digest reactions of
purified
PCR product and plasmid:
PCR product digest reaction (50 μl)
Plasmid digest reaction (50 μl)
Volume
(μl)
Reagent
5
10x CutSmart buffer (NEB, USA)
1.5
Restriction enzyme 1
*
(30 Units)
1.5
Restriction enzyme 2
*
(30 Units)
42
P
urified PCR product
*
Use of
NEB high fidelity (HF) enzymes
is
recommended
2.
Mix
well
and i
ncubate
digests
at 37
̊
C for
1.5
h
3.
Add 2 μl
of 1U/μl
FastAP
Alkaline phosphatase
(Thermo Fisher Scientific, USA)
only
to
the
plasmid digest
to remove 5’ phosphates (prevents self
-
ligation of insert
-
less plasmid
s
)
4.
Incubate
both digests
for another 30 min at 37 ̊C
5.
Mix
digests
with 10 μl
Gel
L
oading
D
ye
, Purple (6x)
(NEB, USA)
6.
Run on a 1%
(w/v)
agarose gel
made up
in 1x TAE buffer
and supplemented with 1x
SYBR
S
afe DNA
Gel S
tain (Thermo Fisher
Scientific
, USA)
for 30 min at 150 V
7.
Excise band
s
with clean razor blade
s
8.
Purify excised DNA band
s
with Zymoclean Gel DNA
R
ecovery
K
it (Zymo Research, USA)
9.
Elute
in two steps
with 2x 10
μl ddH
2
O
(20 μl final volume)
10.
Measure DNA concentration
using a
N
ano
D
rop
spectrophotometer (Thermo Fisher Scientific,
USA)
Ligation
1.
Using the size
in bp
of both digested insert and
plasmid
fragments, calculate the amount of
insert in ng to ligate with 10
0 ng dephosphorylated
plasmid using
a 2:1 insert:plasmid ratio
à
U
se th
e
NEBioCalculator
:
https://nebiocalculator.neb.com/#!/ligation
2.
Set up the ligation reactio
n and include a negative control reaction in which
the
insert is
replaced with
ddH
2
O
Ligation reaction (10 μl)
Volume (μl)
Reagent
5
2
x
Quick
L
igase
Reaction Buffer
X
X ng of insert
DNA
X
100 ng of
plasmid DNA
0.5
Quick
ligase
(NEB, USA
)
Add to
10
ddH
2
O
3.
Mix
well
and incubate for
15
min at room temperature
Transformation
via heat shock
1.
Thaw one 100 μl vial of chemical
ly
competent
E. coli
Stellar cells (Takara Bio, Japan)
per two
ligation reactions for 10 min on ice
2.
Split into 2x 50 μl aliquots in two separate tubes on ice and add 5 μl of ligation reaction to
each
3.
Incubate for 30 min on ice
4.
Heat shock tubes at 42 ̊C for 35 sec in a
thermomixer,
heat block or water bath
5.
Quickly remove to
ice and chill for 1
-
2 min
6.
Rescue by addition of
200
μl SOC recovery medium
Volume
(μl)
Reagent
5
10x CutSmart buffer (NEB, USA)
1.5
Restriction enzyme 1
*
(30 Units)
1.5
Restriction enzyme 2
*
(30 Units)
X
5 μg plasmid DNA
Add to 50
ddH
2
O
7.
Incubate
tubes
at 37 ̊C for 30
-
60 min shaking at 1,200 rpm
8.
Plate out ̃150 μl
on LB
-
Carb
agar
plates, let dry and incubate
upside down
overnight at 37 ̊C
Gibson cloning
Primer
design
Use NEBuilder Assembly tool to design insert primers with matching overhangs for Gibson
assembly:
https://nebuilder.neb.com/#!/
1.
Generate
a
new fragment and copy&pas
te backbone plasmid DNA sequence, click process
text and check the ‘vector’ and ‘circular’ boxes. Rename
‘new fragment’
to ‘plasmid’.
2.
Select ‘restriction digest’ as the method for production of a linearized fragment and specify
your choice of restriction s
ites. Finally click ‘Add’.
3.
Generate
another
new fragment and copy&paste insert template DNA sequence, click
process text and check the ‘vector’ and ‘circular’ boxes if applicable. Rename to ‘insert’.
4.
Select ‘PCR’ as the method for production of a linearize
d fragment and specify the start and
end base of your insert.
Include stop codon if needed.
Finally click ‘Add’.
5.
The exonuclease in the Gibson assembly mix will remove the 4 bp overhangs generated by
restriction digest of the plasmid, leaving only 1 base o
f the original 6 bp recognition site behind.
In order to maintain a proper open reading frame
,
upstream and
/or
downstream junctions
between plasmid and insert fragments
may
need to be adjusted
by adding either two or five
bases, to restore a single codon o
r the complete restriction site, respectively
. Click on
the
pencil symbol of the newly added insert fragment to edit
these junctions
.
6.
The program generates an ‘assembled sequence’ that should be thoroughly inspected to
contain the insert at the correct lo
cation and in the correct reading frame.
7.
If all looks well order the suggested insert primer pair containing the proper Gibson assembly
overhangs.
PCR
amplification of insert fragment
1.
Set up and run PCRs as described above
Restriction
digest
to create plasmid fragment
1.
While the insert PCR is running, set up the plasmid restriction digest reaction as described
above and include the phosphatase treatment step
Gel purification
1.
Mix
insert PCR
and plasmid restriction digest reaction
with 10 μl
Ge
l
L
oading
D
ye
, Purple (6x)
(NEB, USA)
, r
un on a 1% (w/v) agarose and purify from excised gel band
s
as described above
Gibson
assembly
reaction
1.
Calculate the volume of insert
and vector fragment that contains 50 fmoles of each using
NEBioCalculator:
https://nebiocalculator.neb.com/#!/dsdnaamt
2.
Mix calculated volume of both vector a
nd insert fragment and then dilute two
-
fold with 2x
Gibson Assembly Master Mix (NEB,USA)
3.
Incubate at 50 ̊C for 30 min
4.
Transform up to 5 μl into chemical
ly
competent
E. coli
Stellar cells via heat shock as described
above