1. VRIJE UNIVERSITEIT BRUSSEL
INSTITUTE OF MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
TITLE: FINAL ARTICLE PRACTICAL REPORT (SEMESTER 1, 2011)
NAMES: Kariuki S., E. Kamani and A. Garba
INSTRUCTOR: Steven Odongo
DATE OF SUBMISSION: 13/1/2012

2. Recombinant Nanobody™ expression in E. coli, extraction and purification.
Authors names: Kariuki S., E. Kamani and A. Garba.
Institute of Molecular Biology and Biotechnology, Building E, Faculty of Science, Vrije Universiteit Brussel,
Brussels, Belgium.
ABSTRACT:
Nanobody is a fragment antibody consisting of a single monomeric variable antibody domain lacking the
light chains and the CH1 domain of the heavy chain derived from camelides (dromedaries, camels,
Llamas and alpacas). They are less lipophilic and better soluble in water owing to their CDR3 which
forms an extended loop covering the lipophilic site that normally binds to light chains. This property
makes it easier to grow them in bacteria cells and contributes to their therapeutic usefulness.
Escherichia coli WK6 were transformed with pHEN6c containing the nanobody gene with 1M IPTG
(Isopropyl β-D-1 thiogalactopyranoside) to induce expression of the gene and ampicillin-containing LB
media for selection. The cells were harvested by using Beckman Coulter Avanti J-E centrifuge with rotor
JA-10 before lysing through osmotic shock since our protein was expressed in the periplasmic space of
E.coli cells.
Protein purification was done using immobilized metal affinity chromatography (IMAC) with nickel bead
slurry that binds the C-terminal histidine tail tag of our nanobody followed by elution through
competitive action of imidazole buffer to the nickel beads.
Finally the nanobody was analyzed using SDS-PAGE and Western blot. Since SDS-PAGE separates
proteins based primarily on their molecular weight it was possible to determine the molecular weight of
the nanobody. Western blot was used to confirm expression of nanobody in WK6 E.coli by probing with
an antihis-antibody, the 6x histidine tail attached to the C-terminal end of the nanobody.
Key Words: Nanobody, Recombinant, Affinity Chromatography, Western blot, SDS-PAGE,
Coomassie blue.
Abbreviations: HRP, Horse Radish Peroxidase, IPTG,Isopropyl β-D-1-thiogalactopyranoside,
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Agarose Gel Electrphoresis, HIS, Histidine,
BSA, Bovine Serum Albumin, TB, Terrific broth, LB, Luria Broth, IMAC, Immobilized Metal Affinity
Chromatography.

3. 1: INTRODUCTION.
Recombinant protein expression is an extension gene into E.coli plasmid (Cohen, S.; Chang, A.;
of gene expression through transcription, Boyer, H.; Helling, R.,1973). In our experiment,
translation and eventual folding of the protein. E.coli, WK6 was used.
Recombinant proteins show large variability in
terms of their expression, solubility, stability, To achieve a high gene dosage, the cDNA is
and functionality making them difficult targets typically cloned in a plasmid that replicate in a
for large scale production and analyses. relaxed fashion inside a bacteria cell. It is
However greater advancement has been made usually engineered to contain a regulatory
towards solving to improve these features. sequence that act as an enhancer and a
Among them is addition of protein fusion tags promoter region which lead to the efficient
transcription of the gene carried by the
which has improved expression, solubility and
production of biologically active proteins expression vector. In addition a selectable
especially those difficult-to-express-proteins. marker in form of antibiotic resistance, reporter
Genetically engineered tags allow the and a multiple cloning site are required (Amann
purification of the protein without prior E, Brosius J, Ptashne M., 1983). The multiple
knowledge of its biochemical activity (Esposito cloning sites has restriction site with various
D, Chatterjee DK.2006 and Arnau J, Lauritzen C, restriction endonucleases which are molecular
scapels that cut double stranded DNA at
Petersen GE, Pedersen, J.2006). We used a
genetically engineered histidine tag not only for particular recognition nucleotide sequences.
the purification purpose but also in probing These enzymes found in bacteria and archea are
with antihis-antibody. The 6x histidine tag thought to have evolved as defence mechanism
attached to the C-terminal end of the nanobody against vises (Roberts RJ; Murray, Kenneth,
bound to immobilized nickel beads acted as an 1976). The host protects itself by methylation
electron donor thereby detaining the protein in by modification enzyme methylase (Kobayashi
the column and latter eluted by addition of I.,2001). In our experiment the nanobody gene
imidazole which dislodged the histidine tails was ligated to e pHEN6c plasmid which was
used to transform WK6 E.Coli.
from the nickel beads in a competitive fashion.
Escherichia coli is one of the most widely used SDS-PAGE, a technique used to separate
for production of recombinant proteins and its proteins based on their molecular weight was
genetics is the better studied than any other used in analyzing the nanobody. Sodium
microorganism. The understanding of its Dodecyl Sulfate (SDS) is a detergent used to
transcription, translation and gene expression denature the proteins allowing the proteins to
has positioned it as valuable bacterium in exist stably in an extended conformation hence
expression of complex eukaryotic proteins. In they migrate through the pores of the gel
irrespective of their hydrodynamic properties.
addation, E.coli grows rapidly and at high
density in relatively an inexpensive In addition SDS covers all the protein with
media.Expresiion of eukaryotic proteins in negative charges which allows them to migrate
bacteria started with the pioneering work done to the positive electrode. Since polyacrylamide
by Boyer and Cohen when they inserted a frog gel is not solid but rather a meshwork of

4. labyrinth of tunnels, the protein is able to go 2.2 Extraction of the expressed protein
through with the help of electric currents.
After overnight expression, the cells of the WK6
Western blotting was used to confirm E.coli were harvested; 330ml of WK6 E.coli
expression of the nanobody protein. This culture Was centrifuged at 8000rpm for 8
technique detects proteins in minute quantities minutes at the temperature of 140C before
after immobilizing on a gel followed by transfer discarding the supernatant, and centrifugation
to a nitrocellulose membrane. repeated until all cells were harvested. The
pellets were Re-suspended using 12ml Tris
Antibodies and antibody fragments are EDTA sucrose (TES) per pellet, from WK6. E.coli
exclusively applied in human therapy and overnight culture and the mixture incubated for
diagnosis. They are highly specific making them 1 hour on ice while shaking at 200rpm on a
suitable for their uses. However, production of
table shaker. The mixture was supplemented
antibodies via hybridoma technology with 100ml 2M Mgcl2 and centrifuged at
discovered by Cesar Miltein and Georges J. F. 8000rpm for 30 minutes and the periplasmic
Kohler in 1975 (Nelson, PN et all, 2000) remains extract was pipetted into 50ml falcon tube.
expensive and difficult. Camelidae produced a
substancial proportion of their functional 2.3 Protein purifications; Analysis of
immunoglobulins as homodimer of heavy expression and purity of protein sample by
chains, lacking light chains (S. Muyldermans, SDS-PAGE
2001). Since the discovery of camelide antidoby
lacking light chains and CH1 groups domains, The 6x his-tagged Nanobody was purified by
their variable heavy chain domains (VHH) have affinity chromatography,
been proposed as valuable potential tools for HIS-select solution was solubilized to slurry
biotechnology (Hamer C. et all, 1993). and together with periplasmic extract incubated
for 1 hour with shaking. The mixture of
periplasmic extract and HIS-select solution was
2.0 METHODOLOGY loaded onto the column, after which HIS-select
column was washed with 20ml PBS. The PBS
2.1 Expression of Nanobodies in WK6 E.coli buffer was allowed to drain and solution
cells periplasm. collected. The Nanobody was eluted using PBS
WK6 E.coli cells were used for expression of Buffer supplemented with 4ml, 0.5M imidazole
Nanobody. Bacteria were grown at standard and the elute absorbance measured at 280nm.
conditions, at the temperature of 37˚c and The 12% running gel was constituted using
incubated overnight using TB media 4.0ml, 30% Acrylamide/bisacrylamide, 2.5ml
supplemented with ampicillin, glucose and ,1.5M Tris HCl, pH 8.8,3.4ml distilled water,
magnesium chloride in a baffle shaker flask. 100µl 10% SDS,100µl 10% APS, and 5µl TEMED.
IPTG was used to induce expression of the The solution was carefully introduced into gel
Nanobody gene. sandwich, until 0.5cm below the level where
teeth of the comb will reach.1-5mm layer of

5. water was made on top of the separating gel. combs were removed. The gel was placed into
After the gel polymerized the water discarded. electrophoresis chamber and electrophoresis
buffer was added to the inner and outer
Also 4% stacking gel was constituted using 30% reservoir.
0.650 Acrylamide/bisacrylamide, 0.650 1.0M
Tris pH 6.8, 3.645 distilled water, 50µl 10% SDS, The wells were marked 1-6, with different
25µl 10% APS and 5µl TEMED. The stacking gel sample added as; Maker sample, uninduced
was introduced into gel sandwich until solution sample unpurified protein sample, flow through
reached the top of front plate. sample, wash sample, and Eluted sample. 20µl
each of these samples mixed with 5µl of 1x
The combs later were carefully inserted and the sample buffer heated at 100˚c for 5 minutes
gel allowed to polymerize. After which the and added to the wells.
3: RESULTS.
3.1 Expression and protein extraction from WK6 E.coli
Following expression usind IPTG and protein extraction through osmotic shock, the concentration of the
protein was measured using a Nanodrop™ at 280nm and found to be 119µg/µl
3.2 Running Gel And staining with Commasie Blue on SDS-PAGE
The gel was run and then transferred to a small container, containing 20ml commasie blue. The gel was
distained later using commasie distainer, after the bands were visible. Gels were preserved for
molecular weight determination.
MK UI UP FT WS ET
15kDa
10kDa
Figure 1 showing the results of SDS-PAGE, MK is the marker, UI, uninduced sample, UP, unpurified protein, FT, flow through,
WS, wash, ET, elute sample.
3.3 Confirming protein Expression by western-blot and immunodetection.
12% SDS-PAGE Gel was run, and the protein bands transferred from the gel to nitrocellulose membrane
using 35ml transfer buffer.

6. The membrane was transferred to a smaller container, with 8ml 3% PBST buffer. And membrane
blocked using 15ml 1% milk solution to prevent unspecific binding.
Antibodies were poured into the solution of buffer containing the membrane, the membrane washed
with TBS 3 times, and second Antibody which had a HRP conjugate was added and washed again before
adding the developing reagent for HRP and observed for 30minute. The membrane was washed , dried
and scanned.
MK UI UP FT WS ET
15kDa
10kDa
Figure 2, The results of Western blotting after immune-blotting. MK is the marker, UI, uninduced sample, UP, unpurified
protein, WS, wash, ET, elute.
3.3 Calculations for protein molecular weight determination from SDS-PAGE:
The molecular weight of an unknown protein was estimated by comparing its distance of migration
in a gel with that of the standards. A plot of log of the molecular weight (in kDa) of each band of
standard (Y) was done against relative distance traveled from the well (X). A line of best fit was
drawn connecting the points and molecular weight of protein was determined.
Relative distance travelled = Distance of Protein migration from the origin
Distance of migration of dye from the origin

7. Table 1, Showing values of migration fronts (Rf) and log of molecular weight
Relative Molecular Weight of
Migration of standards(cm) distances (X) standard. Log molecular weight (Y)
1.9 0.18 170 2.23
2.1 0.2 130 2.11
2.4 0.23 100 2
2.9 0.28 70 1.85
3.4 0.32 55 1.74
4 0.38 40 1.6
4.7 0.45 35 1.54
5.5 0.52 25 1.4
7.2 0.69 15 1.18
9.1 0.87 10 1
Sample Migration=7.6cm 0.72
Dye migration distance=10.5cm
Protein Molecular Weight determination
2.5
Log of Molecular Weight
2
1.5
1
y = -1.7178x + 2.3727
0.5
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Relative distances
Figure 3 Graph showing the relationship between molecular weight and distance travelled by the protein

8. From the equation of the line;
Y=-1.7178*0.72 + 2.3727
Y=1.135884. Protein molecular weight was obtained by calculating the antilog of 1.135884.
Protein Molecular weight =13.67kDa
4: DISCUSSION.
SDS-PAGE is a technique widely used in molecular biology, biochemistry, forensics, and genetics to
separate proteins according to their electrophoretic mobility, function of length of polypeptide chain or
molecular weight.
We determined the protein molecular weight in this experiment using a plot of log of molecular weight
(kDa) against relative distances on SDS-PAGE using a protein ladder as a standard .The molecular weight
of unknown protein was calculated as shown above indicating that the molecular weight of the protein
is 13.67 kDa .The calculation support with more confidence the prediction of the protein size .SDS PAGE
result shows that the protein was not well purified due to presence of extra small bands together with
the large and thick band of the protein of interest in elute sample.
In this experiment we successfully demonstrated the use of SDS-PAGE for protein purification and
determination of the molecular size of unknown protein sample using the empirical relationship
observed between log of molecular weight (kDa) and relative mobility on SDS-PAGE
Western blot analysis can detect protein of interest from a mixture of a great number of proteins.
Western blotting is useful to give information about the size of your protein with comparison to a size
marker or ladder in kDa, and also on protein expression. In our experiment we used Western blot
technique to confirm expression in WK6 E. coli. The Nanobody expressed contained 6x histidine tags
attached to the C-terminal end. His-tagged Nanobody was identified by probing with anti-his antibody.
The results (Fig 2) above show the Western blot results of different protein samples in different lanes.
On the second lane (UI) from the ladder, (pre induction with IPTG) there were nothing detected which
indicated there were no protein expressed. After induction with IPTG, we had a small band on UP lane
indicating that, there was protein expression. On the ET lane, a heavy band of our protein of interest
was detected and the size, determined using SDS-PAGE was approximately 13.67kDa).The sensitivity of
the assay was high and there were no contaminating bands in our results.
5: CONCLUSION.
In this experiment we were able to employ the use of Western blot and SDS-PAGE to demonstrate
expression of Nanobody containing 6xhistine tail and approximately estimate visually the size by
comparing with the ladder and by mathematical comparison of unknown protein distance of migration
in a gel with that of the standards.

9. 6: ACKNOWLEGMENT.
We acknowledge our practical instructor Steven Odongo for being helpful to us and for his technical
guidance and support during practical training. Not forgetting all the IPMB lecturers who provided us
with the necessary information on Molecular biology.
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