View
12
Download
3
Category
Preview:
Citation preview
Protocols
Experimental Techniques of Molecular
Biology and Protein Expression
Summer term 2017
Cloning and expression of recombinant proteins in E. coli for the development of a
multifunctional membrane for water purification
Student project MultiBrane
Prof. Dr. Budisa
Supervisor: Saba Nojoumi, Franz-Josef Schmitt
1
Microbial cultivations
In this project we only worked with the coliform bacterium Escherichia coli. E. coli is one of
the best studied microorganisms and is commonly used in molecular biology. Naturally, E. coli
occurs in the intestine of mammals as an important part of the gut microbiome. Regularly, E.
coli laboratory strains are regarded as S1 organism, meaning that they are non-pathogenic
and require no special safety measures. Nevertheless, there are also harmful, toxin-producing
E. coli strains and as a scientist working with genetically modified organism we always took
care of an appropriate and sterile working manner.
Figure 1| E. coli cell schematic, source: Aqua di Cutinase project TU Berlin
2
For the cultivation of our E. coli strains we normally used LB medium (liquid broth medium)
that was supplemented with Kanamycin (50 mg/ml). Kanamycin is an antibiotic that is used to
select microorganism. We selected E. coli cells which had uptaken our plasmid and therefore
gathered a kanamycin resistance.
Table 1| LB medium recipe
component [g/L]
Trypton or Pepton 10
Yeast extract 5
NaCl 10
Kanamycin 0.05
dest. water
(adjust pH 7)
add to the desired volume
Agar (for solid media only) 1,5%
Liquid medium was prepared for cultivations of E. coli for transformations or protein
expression. Such cultivations were carried out in small cultivation tubes (10 ml) or in shake
flasks up to 1 L, which were incubated on a shaker platform at 200 rpm and 37 °C for regular
cultivations but 30 °C for protein expression.
Solid medium was prepared in agar plates to streak out E. coli or plating cells
subsequently after transformation to have the ability to select single clones. Incubation was
performed at 37 °C in a static incubator.
For all cultivations, it was taken care to take sterile materials and also work sterile, whenever
possible such work was done under a sterile bench.
3
OD measurement
To monitor growth and culture density we used OD600 measurements. Thereby, the density of
a cell suspension is determined at 600 nm in a spectrometer with cell-free medium as a
reference. The result is an absorbance value that should never exceed 1. If so, the measured
solution must be diluted. We usually measured in a 1 ml cuvette.
4
Competent cells
For the transformation of foreign DNA to E. coli the cells need the ability to take this DNA up.
Such cells are called competent and they need special treatment to increase their membrane
permeability for the uptake.
We applied two different protocols to generate competent cells:
● CaCl2 competent cells (chemically competent)
A 4 ml overnight culture of E. coli cells is used to inoculate 400 ml of fresh LB medium. The
cells are subsequently incubated until an OD600 of maximal 0.7 is reached. The culture is
portioned then into 50 ml reaction tubes and cooled on ice. Next the cells are harvested by
centrifugation at 4 °C for 10 min at a speed of 5000 x g. Cell pellets are cooled on ice and
resuspended in 10 ml of precooled MgCl2 solution (100 mM). The resuspension is chilled on
ice for approx. 30 minutes. Thereafter two aliquots are unified and centrifuged at 4 °C for 10
min at a speed of 4000 rpm. The pellets are resuspended in 2 mL sterile ice-cold CaCl2 solution
(100 mM CaCl2, 15% glycerol) each. The cells are now assumed to be competent.
Competent cells are aliquoted in portions of 50 or 100 μL, frozen in liquid nitrogen and stored
at -80 °C.
● electrocompetent cells
A 2-4 ml overnight culture of E. coli cells is used to inoculate 200 ml of fresh LB medium.
The culture is incubated at 37 °C and 200 rpm until the OD600 reaches 0.3 - 0.5. The cells are
subsequently harvested by centrifugation for 5 min at 4 °C and 3000 x g). The pellet is washed
twice with ice-cold 10% glycerol and afterwards resuspended in ice-cold 10% glycerol to a
final OD600 around 60 – 70. This cell solution is aliquoted in portions of 50 -100 µl, frozen in
liquid nitrogen and stored at -80 °C.
5
DNA amplification with Polymerase chain reaction (PCR)
Polymerase chain reaction, short PCR, has become the common procedure to amplify DNA
in molecular biology. All needed is a DNA template, which contains the desired DNA
sequence, primer - small DNA oligos that match the beginning and the end of the desired DNA
sequence perfectly and a DNA polymerase - an enzyme that can produce copies of DNA.
There are basically 5 steps:
● STEP 1: initial denaturation of the template DNA - to get single strand DNA (otherwise
the DNA polymerase cannot copy the DNA)
● STEP 2: denaturation of the template DNA
● STEP 3: Annealing of the primer
● STEP 4: extension of the primer along the DNA template (DNA copying by DNA
polymerase)
● STEP 5: final elongation - DNA polymerase finishes the copying process for all
templates
These steps are performed at specific temperatures and step 2 - 4 are repeated for several
times which leads to n2 -DNA fragments in each cycle. Optional the reaction can be stopped
and stored at 4 °C for a longer time period in the end.
Table 2| PCR programm
Step Temperature
[°C]
Time [min] Cycle nb.
1 Initial denaturation 95 3:00 1
2 Denaturation 95 0:30 30
3 Annealing 0:30 30
4 Extension 72 30
5 End elongation 72 10:00 1
6 Hold 4 infinity infinity
The annealing temperature depends on the melting temperature of the primers and the
reaction mixture components. It has to be calculated for every PCR which can be done online.
6
The timespan for the extension depends on the size of the DNA fragment and the synthesis
velocity of the applied polymerase.
We worked with the Taq-Polymerase (1 kb/min) and the Q5® High-Fidelity DNA Polymerase
by NEB (2 kb/min). the difference between both polymerases is the synthesis accuracy, which
is higher for the Q5®. Therefore, control PCRs were performed with Taq DNA Polymerase and
amplification of genes for further cloning was performed with Q5® DNA Polymerase.
We prepared a PCR mix freshly for every amplification after the following scheme:
Table 3| PCR mix pipetting scheme
amount comments
DNA template < 1 ng DNA amount needs to be
calculated
polymerase buffer
(10x)
2 µl specific for each polymerase
polymerase 0.125 - 0.25 µl
depends on the polymerase
protocol
primer #1 (10 µM) 0.5 - 1.25 µl
primer #2 (10 µM) 0.5 - 1.25 µl
dNTPs (10 mM) 0.5 µl
nuclease-free water up to 25 µl
Note: The primers were ordered from Sigma-Aldrich after in silico design. After arrival, they
were diluted to 10 µM aliquots and stored at -20 °C.
The result of the PCR is evaluated via gel electrophoresis.
7
Gel electrophoresis
To separate and analyse DNA fragments we used 1% agarose gels. Agarose was boiled with
the running buffer (50x TAE buffer: Tris, acetic acid, 0.5M EDTA) until the solution becomes
completely clear. This solution can be stored at 60 °C. Each gel is prepared with an appropriate
comb and a staining reagent is added. For staining we used either GelRedTM or ethidium
bromide and to determine the DNA fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM
Thermo Fisher). The samples were diluted with nuclease-free water and loading buffer (6x,
Thermo Fisher) to a final volume of 6 µl and loaded to the gel. To run the gel electrophoresis
voltage is applied: we regularly applied 90 V for a 1 h run. However, sufficient separation must
be assured. There are loading buffer systems that include pre-stained DNA fragments (100
bp) which can be monitored during the run for their movement.
The run is stopped by disconnecting the voltage and transferring the gel to a photo-
documentation station. This instrument is used to read out DNA gels as it illuminates the
stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to
ultraviolet light. The exposure can be monitored and captured by a camera. The gel images
can then be analysed.
Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations
even in living organism (mutagenic). The handling of ethidium bromide therefore need special
safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone
and is thus assumed to be non-mutagenic.
8
Gel extraction
To separate DNA fragments of different size and utilization of these in further cloning steps
gel extraction was conducted.
Therefore, the mix that contains the DNA is conditioned with loading buffer and loaded to the
gel. The gel in this case has much larger gel bags thus a large volume can be loaded.
The gel is prestained with either GelRedTM or ethidium bromide and to determine the DNA
fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM Thermo Fisher) is loaded to a small gel
bag. To run the gel electrophoresis voltage is applied: we regularly applied 90 V for a 1 h run.
However, sufficient separation must be assured. There are loading buffer systems that include
pre-stained DNA fragments (100 bp) which can be monitored during the run for their
movement.
The run is stopped by disconnecting the voltage and transferring the gel to a photo-
documentation station. This instrument is used to read out DNA gels as it illuminates the
stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to
ultraviolet light. The exposure can be monitored and captured by a camera. The gel images
can then be analysed.
The exposure of DNA by ultraviolet light induces DNA damage. Since we want to utilize the
DNA from the gel afterwards, the exposure should be short with a low UV light intensity.
Furthermore, this also protects the person working directly at the illumination desk. This
person has to wear UV-protecting glasses and skin protection. The desired DNA fragment is
then cut out of the illuminated gel with a scalpel and transferred to a 1.5 ml reaction tube.
Depending on the applied kit, the reaction tube must be weighed without and with gel pieces
in to determine the weight of these. We used two different Gel extraction kits: Roti®-Prep Gel
Extraction, Roth and GeneJET Gel Extraction Kit, Thermo Scientific. The gel extraction was
performed according to the manufacturers manual. The eluted DNA concentration was
determined by UV - spectroscopy.
Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations
even in living organism (mutagenic). The handling of ethidium bromide therefore need special
safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone
and is thus assumed to be non-mutagenic.
9
DNA concentration measurement
DNA concentration was measured after each step of our cloning protocol. The DNA solution
is measured with a UV-spectrometer in reference to an appropriate buffer blank. In order to
detect protein impurifications and solvent residues, the measurement includes four
wavelengths.
260 nm - DNA
280 nm - proteins
230 nm - organic impurifications
340 nm - ethanol
10
DNA purification
After enzymatic treatment of DNA with either polymerases or restriction enzymes the DNA
needs to be purified from theses enzymes and their buffers. Regularly, we used DNA
purification Kits for that (GeneJET PCR Purification Kit, Thermo Scientific). The purification
was performed according to the manufacturer’s manual.
In case we wanted to monitor the DNA between cloning steps we did a gel purification or gel
extraction.
11
Restriction digest
Restriction enzymes are part of the bacterial immune system and cut DNA specifically. For
this these enzymes need a specific recognition sequence called restriction site. Restriction
sites are mostly palindromic DNA sequences. When such as sequences is cut by a specific
enzyme this can either result in blunt ends without overhangs or sticky ends with overhang.
The enzymes that we applied all create a 5`prime overhang.
To clone our gene of interest we need to cut open the plasmid backbone and make the insert
fit into that frame. For each cloning procedure, we apply two different restriction enzymes to
generate overhangs with different base pairs. Thus, the plasmid cannot repair itself (auto-
ligation).
We prepared a digestion mix freshly for every restriction digest after the following scheme on
ice:
Table 4| Restriction digestion mix pipetting scheme
Volume [µl] comments
DNA up to 1 µg
restriction enzyme buffer 2 (10x buffer) buffer adequate for enzyme
enzyme #1 1 – 1.5 µl When both enzymes are
compatible with the same buffer enzyme #2 1 – 1.5 µl
pure water up to 20 µl
12
The table below presents the restriction sites of each enzyme that we applied. the choice of
enzymes was of course determined by the restriction sites on our plasmid.
Table 5| Restriction enzymes
enzyme restriction site
AgeI (BshTI) 5’...A’CCGGT...3’
3’...TGGCC’A...5’
BamHI 5’...G’GATCC...3’
3’...CCTAG’G...5’
BglII 5’...A’GATCT...3’
3’...TCTAG’A...5’
HindIII 5’...A’AGCTT...3’
3’...TTCGA’A...5’
NdeI 5’...CA’TATG...3’
3’...GTAT’AC...5’
SalI 5’...G’TCGAC...3’
3’...CAGCT’G...5’
XhoI 5’...C’TCGAG...3’
3’...GAGCT’C...5’
13
Sticky end ligation
Since we just apply restriction enzymes that cut the DNA double strand with a 5’ prime
overhang, we can perform sticky end ligation directly to the cut DNA fragments.
The T4 ligase is an enzyme that can join DNA strands. With this reaction, we insert our gene
of interest to the plasmid pET30b_FliC. Besides the avoidance of auto-ligation, the usage of
two different restriction enzymes for the restriction digestion will guarantee the correct
orientation of our GOI in the plasmid.
Figure 2| sticky end ligation scheme
The reaction shall be performed with an excess of insert, to promote the insertion. Therefore,
the equation below is used, it calculates the amount of insert and backbone on a mole bases.
The input of plasmid backbone is always 50 ng.
Equation 1|ligation ratio for insert
To calculate the needed volume of insert one needs to know the concentration of the insert
(after restriction), the excess factor (x) and the length of both DNA fragments (bp).
Note: A ligation control was always prepared. The ligation control monitors the auto-ligation
of the plasmid; thus, no insert is added to this ligation mix.
14
We prepared a ligation mix freshly for every ligation reaction after the following scheme on
ice:
Table 6| Ligation mix pipetting scheme
amount comments
Insert x µl as calculated
Backbone
(pET30b_FliC_MCS)
50 ng volume must be calculated
T4 Ligase buffer (10x)
2 µl
T4 Ligase 0.5 µl
pure water To 20 µl
15
Transformation of E. coli
Given the fact that we had two different types of competent cells the transformation was
performed according to each protocol. A transformation was conducted with 50 µl of
competent cells.
● CaCl2 competent cells (chemically competent)
A 50 µl aliquot is thawed on ice and subsequently maximal 5 µl of the ligation mix is added.
The cells are then chilled for 20 minutes on ice followed by a heat shock of 42 °C for exactly
45 seconds. Afterwards the cells are chilled on ice for three minutes and 750 µl LB medium
(w/o Kanamycin) is added. The cells recover for approx. 30 min at 37 °C and 450 rpm. Finally,
the culture is centrifuged for 1 min at room temperature and 7000 rpm speed, the supernatant
is briefly discarded and the residue volume is used to plate all cells to LB agar plates (with
Kanamycin). These plates are incubated overnight at 37 °C.
Note: In case a mini-prepped plasmid was transformed, only 80 µl of the recovered cells are
plated without any centrifugation.
● electrocompetent cells
A 50 µl aliquot is thawed on ice and pipetted into an electroporation cuvette. Afterwards,
maximal 2 µl of the ligation mix is added. The cuvettes are then transferred to the
electroporator and 1.8 kV are applied to the cell solution. 950 μL LB medium is added to the
transformed cells and the suspension is transferred to a sterile 1.5 mL microfuge tube. For cell
recovery, the culture is incubated at 37 °C and 4500 rpm for approx. 1 h. Finally, the
cells are plated on LB medium agar plates (with Kanamycin) and incubated overnight at 37.
Note: In case a mini-prepped plasmid was transformed, only 80 µl of the recovered cells are
plated without any centrifugation.
The plates were monitored the next day and the cfu (colony forming units) were determined.
Additionally, some clones were picked to perform colony PCR and to produce a sorted
backup plate of these clones.
16
Colony-PCR
The success of transformation and ligation was evaluated by colony-PCR. This method is
designed for high-throughput analysis of multiple clones.
The cells are picked from the transformation plate with a sterile pipette tip or a sterile
toothpick and each transferred to 50 µl TE-buffer or pure water. This solution is then boiled
for 10 minutes at 99 °C to lyse the cells and access the DNA (in a heat block).
Meanwhile the PCR reaction mix can be prepared according to the pipetting scheme without
DNA - this is called mastermix. The applied primers are chosen to specifically bind the
plasmid outside the cloning site. Therefore, the size of the amplific
ate will increase if the GOI is inserted.
The variables of the PCR program are then adapted to the chosen primer and the maximal
amplification length (with GOI inserted).
Table 7| PCR mix pipetting scheme for Colony-PCR
amount comments
DNA template 1 µl from boiled cell solution
Taq polymerase buffer
(10x)
2 µl for Taq Polymerase
Taq Polymerase 0.6 µl
according to Taq Polymerase
protocol
primer #1 (10 µM) 0.5 µl
primer #2 (10 µM) 0.5 µl
dNTPs (10 mM) 0.5 µl
nuclease-free water up to 20 µl
The reaction mix can be upscaled to the number of clones that should be analyzed and
prepared without the DNA template (master mix). This reaction mix can be distributed to PCR
reaction tubes and subsequently 1 µl of the boiled cell solution is added.
17
Table 8| PCR program for Colony-PCR
Step Temperature
[°C]
Time [min] Cycle nb.
1 Initial denaturation 95 3:00 1
2 Denaturation 95 0:30 30
3 Annealing 0:30 30
4 Extension 72 30
5 End elongation 72 10:00 1
6 Hold 4 infinity infinity
The annealing temperature depends on the melting temperature of the primers and the
reaction mixture components. It has to be calculated for every PCR which can be done online.
The timespan for the extension depends on the size of the DNA fragment. The synthesis
velocity of Taq Polymerase is 1 kb/min.
18
Miniprep
To isolate plasmid DNA from E. coli cells, when they were approved by colony-PCR or needed
to be transformed to another strain, was performed with the Thermo Fisher Miniprep Kit. All
steps are conducted as described in the manufacturers manual.
The amount of DNA eluted was evaluated always by UV - spectroscopy.
19
Sequencing
To identify or verify correct sequences of plasmid DNA we used DNA sequencing. First,
DNA concentration is determined by UV spectrophotometry. 12 μL of approx. 70 ng
of plasmid DNA are mixed with 3 μL of sequencing primer at a final primer
concentration of 2 μM. The primers are chosen as such that read lengths of 1 kb is not
exceeded. Plasmid DNA with higher concentration is diluted with
nuclease free water before to prepare approx. 70 ng sequencing sample. The
premixed samples were sent to Seqlab for sequencing.
20
Part II
Protein expression and purification
21
Expression systems background
T7-polymerase dependent expression
For protein expression the fusion-protein constructs are transformed to a different E. coli
strain: E. coli BL21gold. Since the recombinant protein should be produced in a high
amount, this protein expression strain is equipped with the T7-polymerase controlled by the
Lac-Operon.
This DNA dependent RNA-polymerase originally comes from the T7 bacteriophage. A phage
specifically attacks bacteria and replicates itself within the bacterial cells. This works as such
that the phage brings its own gene transcription system to specifically transcribe only its
genes and none from the the host cell, which will then be translated to proteins. This system
is a useful tool of genetic engineering as is guarantees the cells to produce predominantly
the desired protein when the T7-promoter-terminator systems is als present. Since we do not
want protein expression before we checked the plasmid, the cloning strain (E. coli DH10B)
was not equipped with the T7-polymerase. When our plasmid with the T7-promoter and T7-
terminator is transformed to our expression strain, expression would occur immediately.
However, recombinant protein expression is a huge burden for the cells and can induce
rapid cell death. To generate biomass before protein expression the production of proteins is
inducible. This is realized with the Lac-Operon. The Lac-Operon can inhibit and activate
gene expression dependent on Lactose or Lactose-analogues such as IPTG (Isopropyl-β-D-
thiogalactopyranosid).
When IPTG is added to a culture of E. coli BL21gold the T7-polymerase gene can be
transcribed and subsequently transcribe multiple copies of our gene of interest (Fig. 3).
22
Figure 3| T7-expression systems with IPTG induction, Patent Host-vector system for cloning and expressing genes. US
8623652 B2, 2014.
23
Protein Expression with selective pressure incorporation (SPI)
For protein expression in combination with selective pressure incorporation the cloned
plasmids are transformed to a different E. coli. strain: E. coli. B834. Since the recombinant
protein should be produced in a high amount, this protein expression strain is equipped with
the T7-polymerase controlled by the Lac-Operon. This system is similar to the expression of
fusion protein and is explained in detail in the fusion protein expression section.
Selective pressure incorporation is a method that enables scientists to incorporate non-
canonical amino acids (ncAA) in the amino acid sequence of protein. This method is used
predominantly in the field of structural biology but also in Xenobiology and facilitates
research regarding protein structure, stability and function (Budisa et al., 1998). However, it
also enables to incorporate amino acids which are capable of forming a covalent bond
besides the peptide bond enabling biomolecular click-chemistry (Kolb et al., 2001; Rösner et
al., 2015). SPI can take place because there is a naturally “relaxed” substrate specificity of
the Aminoacyl tRNA synthetase (aaRS) to the corresponding amino acid. That means the
affinity of the aaRS to the amino acid is under kinetic control and there is the possibility to
incorporate ncAAs.
Figure 4| Schematic description of the reprogramming of the protein translational machinery; Budisa.2005.
Engineering the Genetic Code. Wiley Weinheim, 1st Edition
Since the affinities of the canonical amino acids (cAA) are way higher than of any ncAA, SPI
doesn’t occur besides the E. coli strain is a knockout strain for the particular AA one wants to
replace (Rubini, 2004). Those strains were modified to have lost the capability of producing
24
one or more amino acids themselves, thus enabling them to take up an artificial analogue and
load it to the respective t-RNA (Rubini, 2004; Budisa et al., 1998). Since, it is desired to
maintain the protein’s functionality it is very promising to use methionine-auxotrophic strains
as methionine does not mediate any functional properties and is a relatively uncommon AA in
comparison to the other canonical AA (Rubini, 2004; Budisa et al., 1997). Thus, abundance
plays a crucial role too.
E. coli B834 also is a methionine-auxotrophic strain and incorporates azidohomoalanine (Aha)
or homopropargylglycine (Hpg) which have structural similarities to methionine.
The different affinity and therefore kinetic rates for the incorporation of ncAAs are depictured
in figure 5.
Figure 5| Lecture: Biotransformation und Synthetische Biologie, SoSe 2016, Technical University of Berlin, 2016.
25
For selective pressure incorporation this methionine-auxotrophic strain is cultured in
absence of methionine but with either Aha or Hpg (Fig. 6).
Both artificial amino acids have a reactive side chain, Aha - azide, Hpg - alkyne, to form a
covalent azide-alkyne bond via click chemistry. That will be used for coupling the root
flagellins to our cellulose backbone.
Figure 6| Incorporation of Aha while protein synthesis, Rösner et al. 2015.Click chemistry for targeted protein ubiquitylation and
ubiquitin chain formation. Nature Protocols 10, 1594–1611 (2015)
26
Induced Expression with E. coli
The basis for each expression is an overnight culture in adequate LB-medium (selective
pressure towards the plasmid by e.g. Kanamycin, see microbial cultivation section). The next
day a novel culture flask with fresh appropriate media is inoculated with 1 ml of the overnight
culture. This culture is monitored by OD600 determination at intervals until an OD600 of 0.5 –
0.7. The culture is then ready for production since the cells are in the exponential growth phase
and we have a sufficient number of cells. Production of recombinant proteins is an additional
burden to the host cells; thus, the culture is induced for production after some time of unlimited
growth. Before induction a SDS-Page sample was taken (see SDS-Page section).
Induction is performed with 0.8 mM IPTG (Isopropyl-β-D-thiogalactopyranosid, 1 M stock).
Furthermore, we occasionally tested expression with the addition of 0.2% lactose to the
culture.
The induced culture is then cultivated for 16 h at 20 °C and 180 rpm. These conditions were
found to be desirable to avoid the formation of inclusion bodies in E. coli. E. coli is unable to
secret proteins into the medium and if a critical level of proteins accumulates in the cells they
form aggregates referred to as inclusion bodies. Recovery functional proteins from inclusion
bodies is laborious and often not very successful.
After 16 h the OD600 is measured again, another SDS-Page sample is taken, and the culture
is subsequently harvested by centrifugation (4 °C, 8000 x g, 10 min).
A SDS-Page sample was taken from the supernatant, which was then discarded. The cells
were resuspended 15 ml NA-buffer (see purification section) and taken to cell lysis. When not
processed immediately, the cells can also be stored at – 20 °C.
When selective pressure incorporation (SPI) is performed alongside with the protein
expression the conditions are different.
The basis still is a culture, of a methionine-auxotrophic strain (E. c. B834) respectively, grown
in LB (with Kanamycin) at 37 °C,200 rpm for approx. 8 h. The main culture is performed in
NMM media (methionine free, with Kanamycin, see recipe below). Only a very low amount of
methionine is yet added to first induce the protein synthesis machinery of the cells and this
culture is incubated overnight at 30 °C,200 rpm. The next day the culture is checked for an
OD600 to be around 0.5 – 0.7. The SDS-Page sample is taken, and the culture subsequently
induced with 500 µl/l IPTG. Additionally, the methionine analogue is now added to the medium:
Homopropargylglycine 50 mg/l or Azidohomoalanine 100 mg/l). Expression is conducted for
3-4 h at 28 °C. The following steps are similar to the main protocol above.
27
Table 9| Composition of NMM medium from stock to final concentration for each component for 1 – 10 l. Ampicillin
is just a representative for antibiotics and is replaced for Kanamycin in our experiments.
28
UV-spectroscopy
For the determination of the concentration of our proteins we used UV-spectroscopy since
proteins absorb specifically at 280 nm. For the measurement a 1:10 solution in the
respective protein buffer is made and this sample is measures against a blank which only
contains the similar buffer but no protein. Classically, the protein concentration is then
measured by the correlation of Lambert-Beers Law (Eq. 2). Since the molecular extinction
coefficient is essential in that context and can vary from buffer to buffer we regularly used
the software tool ProtParam (Gesteiger et al., 2005) which calculates another absorbance
value in silico A0.1%. This value can be used to calculate the protein concentration [mg/ml] by
simple division of A / A0.1%.
𝐴 = ℰ ∗ 𝑑 ∗ 𝑐
Equation 2| Lambert-Beers-Law, A = absorbance value, Ɛ = molar extinction coefficient [l/cm*mol or g](depends
on sample and wavelength) , d = thickness of cuvette [cm] (often 1 cm), c = concentration of analyte [mol/l or g/l]
29
Affinity chromatography
Since all our proteins were cloned with a His-Tag, they can be separated from the other
proteins of E. coli by affinity chromatography. Nickel has a high affinity for Histidine and is
therefore applied as immobilized ligand in the column (IMAC = immobilizes metal ion affinity
chromatography). We used Ni-NTA columns from GE Healthcare (HisTrap™) for either 1 ml
or 5 ml.
For the purification we used different buffers, which are listed in the table below (table 10).
The workflow is also outlined in table format (table 11). The parts of sample treatment are to
be repeated for each sample and the elution is supervised by a PDA-detector (photodiode
array detector) that measures the flow as it passes for its absorbance at 280 nm.
Table 10| Composition of buffers required for affinity chromatography and related steps.
Buffer Amount Component
NA buffer (pH 8)
50 mM NaH2PO4
300 mM NaCl
20 mM Imidazole
5% Glycerol (optional)
NB buffer (pH 8)
50 mM NaH2PO4
300 mM NaCl
40 mM Imidazole
5% Glycerol (optional)
NC buffer (pH 8)
50 mM NaH2PO4
300 mM NaCl
300 mM Imidazole
5% Glycerol (optional)
30
Lysis buffer (pH 8) 50 mM NaH2PO4
500 mM NaCl
25 mM Imidazole
0.05% Tween 20
Wash buffer (pH 8)
50 mM NaH2PO4
500 mM NaCl
500 mM Imidazole
0.05% Tween 20
Wash buffer (pH 8)
50 mM NaH2PO4
500 mM NaCl
10 mM Imidazole
0.05% Tween 20
TEV reaction buffer (pH 8)
50 mM NaH2PO4
0.5 mM EDTA
1 mM DTT
Storage buffer (pH 8)
50 mM NaH2PO4
100 mM NaCl
5% Glycerol
31
Table 11| Workflow at LC-system (liquid chromatography) for His-tagged proteins.
Step In column Column
volumes
Out of column
Pretreatment Wash dH2O 5 Waste
Wash NC buffer 5 waste
Sample
treatment
Conditioning NA buffer 5 Waste
Load sample All cell lysate Collect flow
Wash 1 NB buffer 3 Collect wash 1
Wash 2 NC buffer Until elution
starts
Collect wash 2
Elution NC buffer Until elution
ends
Eluate
Cleaning NC buffer 2-3 Waste
Column
recovery
Wash dH2O 5 Waste
Storage 5 cv 20 5-10 waste
Prior to every purification we lysed the cells by adding lysis buffer and using a homogenizer
for > 10 pump steps with 60 Psi.
v
32
Dialysis
To change the buffer of our purified enzymes we used dialysis. This method in performed with
semipermeable membranes that allow molecules of a certain size to pass and therefore
change the buffer conditions of the proteins inside the membrane. The driving force for this
exchange is diffusion to equilibrate the concentrations of buffer components.
We apply dialysis membranes form Roth with a molecular cut off of 12- 14 kDa (Membra-
Cel™, Roth). The protein in buffer is filled into the closed membrane bag, which is placed into
a much higher volume (1 – 500fold) of the buffer that the protein should be in afterwards.
Dialysis is usually performed overnight or just a couple of hours, that depends on the initial
volume of the sample.
Regularly, we exchanged Nc-buffer and TEV buffer (4 °C, overnight) as well as TEV buffer and
NA buffer (4 °C, overnight) or NA buffer and storage buffer (4 °C, overnight) as.
33
TEV digest
After the initial purification of the His-tagged enzymes the His-tag needs to be cleaved off,
since, it hinders protein folding and functionality. Therefore, we use the TEV protease
(Tobacco Etch Virus protease) an endopeptidase that specifically cleaves ENLYFQ\S (amino
acid sequence) and releases the proteins from their His-tag.
The buffer needs to be exchanged from Nc-buffer to TEV buffer by dialysis (see dialysis
section). When the proteins are in the appropriate buffer, the concentration is measured by
VIS-spectroscopy (see VIS-spectroscopy section).
The protease is then added to the protein solution according to the following equation (Eq. 2)
and the sample is incubated at room temperature overnight.
𝑉𝑇𝐸𝑉 = 1
𝑐𝑇𝐸𝑉∗
𝑚𝑝𝑟𝑜𝑡𝑒𝑖𝑛
100
Equation 3: Formula to take a volume of OD600 = 1, dependent on the measured OD600 value. This makes the SDS-
Page analysis more comparative.
After TEV digestion, the desired enzymes are separated from the cut-off His-tags by IMAC
affinity chromatography. The protocol is basically the same, but now the proteins are within
the flow through when the column is loaded (see modified table below). That can be seen
also with the PDA-detector (280 nm).
34
Table 12| Workflow at LC-system (liquid chromatography) for protein purification from His-tag.
Step In column Column
volumes
Out of column
Pretreatment Wash dH2O 5 Waste
Wash NC buffer 5 waste
Sample
treatment
Conditioning NA buffer 5 Waste
Load sample All cell lysate Collect flow
(protein of
interest)
Wash NA buffer Until no signal
by detector
Collect flow
(protein of
interest)
Cleaning/Elution NC buffer 2-3 Waste
Column
recovery
Wash dH2O 5 Waste
Storage 5 cv 20 5-10 waste
35
SDS-Page
SDS-Page stands for sodiumdodecylsulfate polyacrylamid gel electrophoresis and represents
the counterpart for proteins of agarose gel electrophoresis. It is a simple and fast method to
pretest our expression for the desired protein. The identification is, similar to agarose gel
electrophoresis, by size only since the proteins are linearized by SDS.
Furthermore, we used this method to monitor the efficiency and success of our protein
expression strength, recovery and purification steps. Therefore, we took the following samples
via the process of each protein production:
• sample of culture before induction (S1, volume calculated)
• sample of culture at the end of induced expression (S2, volume calculated)
• sample of supernatant after cell harvesting (S3 - optional)
• sample of supernatant after cell lysis (S4, 50 µl)
• sample of cells debris after cell lysis (S5, 50 µl)
• sample of the column flow through (S6, 50 µl)
• sample of the washing steps (S7, 50 µl)
• sample of the eluate (S8, 15 µl)
Those samples taken directly from culture broth were calculated to equal an OD600 of 1 by the
following formula:
𝑉𝑠𝑎𝑚𝑝𝑙𝑒𝑠 =1000
𝑐𝑢𝑙𝑡𝑢𝑟𝑒′𝑠 𝑂𝐷600
Equation 4| Formula to take a volume of OD600 = 1, dependent on the measured OD600 value. This makes the SDS-
Page analysis more comparative.
The samples were all centrifuges at maximum speed of a desktop centrifuge for approx. 1 min
and the supernatant was discarded. Cells were resuspended in 60 µl pure water and 15 µl of
SDS-buffer. The buffer composition is given below. This solution was boiled than at 96 °C for
10 min and could then be loaded to the gel.
36
Table 13| Components of the 5x SDS-buffer
Amount Component
5x SDS buffer
(ready to boil and
subsequently load samples)
80 mM Tris pH 6.8
10% SDS
12.5% Glycerin
4% (v/v) 2-Mercaptoethanol
0.2% (w/v) Bromophenol blue
We prepared the SDS-gels, containing stacking and separation gel, ourselves according to
the following protocol:
Table 14| Recipe for preparation of SDS-Page gels and running buffer.
Gel type Amount Component
Stacking gel 125 mM stacking gel buffer
(1.5 M Tris-HCl pH 8.8)
5% Acrylamide
0.1% SDS
0.05% APS
0.17% TEMED
Separation gel 380 mM separation gel buffer
(0.25 M Tris-HCl pH 6.8)
12% Acrylamide
0.1% SDS
0.05% APS
0.05% TEMED
37
SDS-running buffer (1L) 30 g Tris-base
144 g Glycine
10 g SDS
Fill to 1000 ml
adjust pH 8.3
water
Before the samples were loaded to the gel, the gels were arranged in the gel chamber with
the running buffer and all bags were rinsed with a syringe. For the analysis of the gel a protein
ladder is added which must be chosen properly for the mass range the analyte is in. We usually
used the PageRuler™ (Thermo Scientific). Then, 10 µl of each sample is loaded and the gel
is run initially at 80 V for 30 minutes to compile all molecules to the running front. Afterwards,
the voltage in increased to 180 V for approx. 60 min.
The gel is subsequently stained with Coomassie staining, covered by the solution for 30 min
at room temperature. Thereafter, the gels are destained in water overnight while being slowly
shaked. The analysis is performed with a photo-dokumentation system.
38
Click reaction of membrane linker with root flagellin
For the functionalization of the membrane, the membrane first has is provided with a root
flagellin. Functionalized flagella fusion proteins can subsequently assemble onto the root to
form a flagella polymer. Both the membrane and the root flagellin have either an alkine or an
azide functional group from the non-canonic amino acids that were incorporated (Aha, Hpg).
For the click reaction, the following solutions were prepared:
Table 15| Recipe of click reaction buffer. Please note that the given concentrations refer to the amount of the
compound that should be added to the solution and does not mean a final concentration.
amount to be added
Potassium phosphate buffer (pH 7.6)
Potassium phosphate (K2HPO4, KH2PO4) 100 mM
NaCl 100 mM
Further components
amino guanidine hydrochloride 100 mM
THPTA 50 mM
CuSO4 20 mM
1:2 CuSO4:THPTA
sodium ascorbate 100 mM
The chemicals were diluted in potassium phosphate buffer. Sodium ascorbate is unstable in
aqueous solutions thus, it can only be diluted right before the reaction starts.
In a 50 ml round bottom flask, 10 ml of potassium phosphate buffer is mixed with a piece of
cellulose acetate with the respective linker, 1 mg FliC_NtermAHA/ HPG (the counterpart to
the linker on the membrane), aminoguanidine hydrochloride, sodium ascorbate, 1:2
CuSO4:THPTA. The air in the flask was replaced by N2 and the flask sealed with a septum. A
balloon was also filled with N2 and connected to the flask by a cannula through the septum to
maintain the N2 saturation in the flask.
Under a fume hood, the reaction mixture is stirred over night at room temperature.
When finished, the membrane was washed tree times in potassium phosphate buffer.
39
Flagella assembly
The flagellin monomers can be assembled either on root flagellin linked by click chemistry to
the cellulose membrane, or in solution. We performed the assembly on the membrane in a
24-Well plate. The flagellin (fusion) proteins have a start concentration of 4 mg/ml and are
diluted in assembly buffer. Finally, the solution has to cover the whole membrane (~ 450 µl
in a 24-Well plate).
To start the flagella assembly, 0.4M of (NH4)2SO4 is added and the sample is incubated at
28°C for 4 hours.
Table 16| Recipe of assembly buffer for flagella assembly
concentration
assembly buffer (pH 6.5)
NaH2PO4 40 mM
NaCl 30 mM
Recommended