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

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.

1: INTRODUCTION.

Recombinant protein expression is an extension

of gene expression through transcription,

translation and eventual folding of the protein.

Recombinant proteins show large variability in

terms of their expression, solubility, stability,

and functionality making them difficult targets

for large scale production and analyses.

However greater advancement has been made

towards solving to improve these features.

Among them is addition of protein fusion tags

which has improved expression, solubility and

production of biologically active proteins

especially those difficult-to-express-proteins.

Genetically engineered tags allow the

purification of the protein without prior

knowledge of its biochemical activity (Esposito

D, Chatterjee DK.2006 and Arnau J, Lauritzen C,

Petersen GE, Pedersen, J.2006). We used a

genetically engineered histidine tag not only for

the purification purpose but also in probing

with antihis-antibody. The 6x histidine tag

attached to the C-terminal end of the nanobody

bound to immobilized nickel beads acted as an

electron donor thereby detaining the protein in

the column and latter eluted by addition of

imidazole which dislodged the histidine tails

from the nickel beads in a competitive fashion.

Escherichia coli is one of the most widely used

for production of recombinant proteins and its

genetics is the better studied than any other

microorganism. The understanding of its

transcription, translation and gene expression

has positioned it as valuable bacterium in

expression of complex eukaryotic proteins. In

addation, E.coli grows rapidly and at high

density in relatively an inexpensive

media.Expresiion of eukaryotic proteins in

bacteria started with the pioneering work done

by Boyer and Cohen when they inserted a frog

gene into E.coli plasmid (Cohen, S.; Chang, A.;

Boyer, H.; Helling, R.,1973). In our experiment,

E.coli, WK6 was used.

To achieve a high gene dosage, the cDNA is

typically cloned in a plasmid that replicate in a

relaxed fashion inside a bacteria cell. It is

usually engineered to contain a regulatory

sequence that act as an enhancer and a

promoter region which lead to the efficient

transcription of the gene carried by the

expression vector. In addition a selectable

marker in form of antibiotic resistance, reporter

and a multiple cloning site are required (Amann

E, Brosius J, Ptashne M., 1983). The multiple

cloning sites has restriction site with various

restriction endonucleases which are molecular

scapels that cut double stranded DNA at

particular recognition nucleotide sequences.

These enzymes found in bacteria and archea are

thought to have evolved as defence mechanism

against vises (Roberts RJ; Murray, Kenneth,

1976). The host protects itself by methylation

by modification enzyme methylase (Kobayashi

I.,2001). In our experiment the nanobody gene

was ligated to e pHEN6c plasmid which was

used to transform WK6 E.Coli.

SDS-PAGE, a technique used to separate

proteins based on their molecular weight was

used in analyzing the nanobody. Sodium

Dodecyl Sulfate (SDS) is a detergent used to

denature the proteins allowing the proteins to

exist stably in an extended conformation hence

they migrate through the pores of the gel

irrespective of their hydrodynamic properties.

In addition SDS covers all the protein with

negative charges which allows them to migrate

to the positive electrode. Since polyacrylamide

gel is not solid but rather a meshwork of

labyrinth of tunnels, the protein is able to go

through with the help of electric currents.

Western blotting was used to confirm

expression of the nanobody protein. This

technique detects proteins in minute quantities

after immobilizing on a gel followed by transfer

to a nitrocellulose membrane.

Antibodies and antibody fragments are

exclusively applied in human therapy and

diagnosis. They are highly specific making them

suitable for their uses. However, production of

antibodies via hybridoma technology

discovered by Cesar Miltein and Georges J. F.

Kohler in 1975 (Nelson, PN et all, 2000) remains

expensive and difficult. Camelidae produced a

substancial proportion of their functional

immunoglobulins as homodimer of heavy

chains, lacking light chains (S. Muyldermans,

2001). Since the discovery of camelide antidoby

lacking light chains and CH1 groups domains,

their variable heavy chain domains (VHH) have

been proposed as valuable potential tools for

biotechnology (Hamer C. et all, 1993).

2.0 METHODOLOGY

2.1 Expression of Nanobodies in WK6 E.coli

cells periplasm.

WK6 E.coli cells were used for expression of

Nanobody. Bacteria were grown at standard

conditions, at the temperature of 37˚c and

incubated overnight using TB media

supplemented with ampicillin, glucose and

magnesium chloride in a baffle shaker flask.

IPTG was used to induce expression of the

Nanobody gene.

2.2 Extraction of the expressed protein

After overnight expression, the cells of the WK6

E.coli were harvested; 330ml of WK6 E.coli

culture Was centrifuged at 8000rpm for 8

minutes at the temperature of 140C before

discarding the supernatant, and centrifugation

repeated until all cells were harvested. The

pellets were Re-suspended using 12ml Tris

EDTA sucrose (TES) per pellet, from WK6. E.coli

overnight culture and the mixture incubated for

1 hour on ice while shaking at 200rpm on a

table shaker. The mixture was supplemented

with 100ml 2M Mgcl2 and centrifuged at

8000rpm for 30 minutes and the periplasmic

extract was pipetted into 50ml falcon tube.

2.3 Protein purifications; Analysis of

expression and purity of protein sample by

SDS-PAGE

The 6x his-tagged Nanobody was purified by

affinity chromatography,

HIS-select solution was solubilized to slurry

and together with periplasmic extract incubated

for 1 hour with shaking. The mixture of

periplasmic extract and HIS-select solution was

loaded onto the column, after which HIS-select

column was washed with 20ml PBS. The PBS

buffer was allowed to drain and solution

collected. The Nanobody was eluted using PBS

Buffer supplemented with 4ml, 0.5M imidazole

and the elute absorbance measured at 280nm.

The 12% running gel was constituted using

4.0ml, 30% Acrylamide/bisacrylamide, 2.5ml

,1.5M Tris HCl, pH 8.8,3.4ml distilled water,

100µl 10% SDS,100µl 10% APS, and 5µl TEMED.

The solution was carefully introduced into gel

sandwich, until 0.5cm below the level where

teeth of the comb will reach.1-5mm layer of

water was made on top of the separating gel.

After the gel polymerized the water discarded.

Also 4% stacking gel was constituted using 30%

0.650 Acrylamide/bisacrylamide, 0.650 1.0M

Tris pH 6.8, 3.645 distilled water, 50µl 10% SDS,

25µl 10% APS and 5µl TEMED. The stacking gel

was introduced into gel sandwich until solution

reached the top of front plate.

The combs later were carefully inserted and the

gel allowed to polymerize. After which the

combs were removed. The gel was placed into

electrophoresis chamber and electrophoresis

buffer was added to the inner and outer

reservoir.

The wells were marked 1-6, with different

sample added as; Maker sample, uninduced

sample unpurified protein sample, flow through

sample, wash sample, and Eluted sample. 20µl

each of these samples mixed with 5µl of 1x

sample buffer heated at 100˚c for 5 minutes

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

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.

15kDa

10kDa

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

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

15kDa

10kDa

Table 1, Showing values of migration fronts (Rf) and log of molecular weight

Migration of standards(cm) Relative distances (X)

Molecular Weight of 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

Figure 3 Graph showing the relationship between molecular weight and distance travelled by the protein

Protein Molecular Weight determination

y = -1.7178x + 2.3727

0

0.5

1

1.5

2

2.5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Relative distances

Lo

g o

f M

olecu

lar W

eig

ht

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.

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.

7: REFERENCES.

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