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  • 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 thelight 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 whichforms an extended loop covering the lipophilic site that normally binds to light chains. This propertymakes 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 LBmedia for selection. The cells were harvested by using Beckman Coulter Avanti J-E centrifuge with rotorJA-10 before lysing through osmotic shock since our protein was expressed in the periplasmic space ofE.coli cells.Protein purification was done using immobilized metal affinity chromatography (IMAC) with nickel beadslurry that binds the C-terminal histidine tail tag of our nanobody followed by elution throughcompetitive action of imidazole buffer to the nickel beads.Finally the nanobody was analyzed using SDS-PAGE and Western blot. Since SDS-PAGE separatesproteins based primarily on their molecular weight it was possible to determine the molecular weight ofthe nanobody. Western blot was used to confirm expression of nanobody in WK6 E.coli by probing withan 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 interms of their expression, solubility, stability, To achieve a high gene dosage, the cDNA isand functionality making them difficult targets typically cloned in a plasmid that replicate in afor large scale production and analyses. relaxed fashion inside a bacteria cell. It isHowever greater advancement has been made usually engineered to contain a regulatorytowards solving to improve these features. sequence that act as an enhancer and aAmong them is addition of protein fusion tags promoter region which lead to the efficient transcription of the gene carried by thewhich has improved expression, solubility andproduction of biologically active proteins expression vector. In addition a selectableespecially those difficult-to-express-proteins. marker in form of antibiotic resistance, reporterGenetically engineered tags allow the and a multiple cloning site are required (Amannpurification of the protein without prior E, Brosius J, Ptashne M., 1983). The multipleknowledge of its biochemical activity (Esposito cloning sites has restriction site with variousD, Chatterjee DK.2006 and Arnau J, Lauritzen C, restriction endonucleases which are molecular scapels that cut double stranded DNA atPetersen GE, Pedersen, J.2006). We used agenetically engineered histidine tag not only for particular recognition nucleotide sequences.the purification purpose but also in probing These enzymes found in bacteria and archea arewith antihis-antibody. The 6x histidine tag thought to have evolved as defence mechanismattached 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 methylationelectron donor thereby detaining the protein in by modification enzyme methylase (Kobayashithe column and latter eluted by addition of I.,2001). In our experiment the nanobody geneimidazole 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 separatefor production of recombinant proteins and its proteins based on their molecular weight wasgenetics is the better studied than any other used in analyzing the nanobody. Sodiummicroorganism. The understanding of its Dodecyl Sulfate (SDS) is a detergent used totranscription, translation and gene expression denature the proteins allowing the proteins tohas positioned it as valuable bacterium in exist stably in an extended conformation henceexpression 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 highdensity in relatively an inexpensive In addition SDS covers all the protein withmedia.Expresiion of eukaryotic proteins in negative charges which allows them to migratebacteria started with the pioneering work done to the positive electrode. Since polyacrylamideby 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 proteinthrough with the help of electric currents. After overnight expression, the cells of the WK6Western blotting was used to confirm E.coli were harvested; 330ml of WK6 E.coliexpression of the nanobody protein. This culture Was centrifuged at 8000rpm for 8technique detects proteins in minute quantities minutes at the temperature of 140C beforeafter immobilizing on a gel followed by transfer discarding the supernatant, and centrifugationto a nitrocellulose membrane. repeated until all cells were harvested. The pellets were Re-suspended using 12ml TrisAntibodies and antibody fragments are EDTA sucrose (TES) per pellet, from WK6. E.coliexclusively applied in human therapy and overnight culture and the mixture incubated fordiagnosis. They are highly specific making them 1 hour on ice while shaking at 200rpm on asuitable for their uses. However, production of table shaker. The mixture was supplementedantibodies via hybridoma technology with 100ml 2M Mgcl2 and centrifuged atdiscovered by Cesar Miltein and Georges J. F. 8000rpm for 30 minutes and the periplasmicKohler in 1975 (Nelson, PN et all, 2000) remains extract was pipetted into 50ml falcon tube.expensive and difficult. Camelidae produced asubstancial proportion of their functional 2.3 Protein purifications; Analysis ofimmunoglobulins as homodimer of heavy expression and purity of protein sample bychains, lacking light chains (S. Muyldermans, SDS-PAGE2001). Since the discovery of camelide antidobylacking light chains and CH1 groups domains, The 6x his-tagged Nanobody was purified bytheir variable heavy chain domains (VHH) have affinity chromatography,been proposed as valuable potential tools for HIS-select solution was solubilized to slurrybiotechnology (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 PBS2.1 Expression of Nanobodies in WK6 E.coli buffer was allowed to drain and solutioncells periplasm. collected. The Nanobody was eluted using PBSWK6 E.coli cells were used for expression of Buffer supplemented with 4ml, 0.5M imidazoleNanobody. 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 usingincubated overnight using TB media 4.0ml, 30% Acrylamide/bisacrylamide, 2.5mlsupplemented 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 gelNanobody 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 intoAfter the gel polymerized the water discarded. electrophoresis chamber and electrophoresis buffer was added to the inner and outerAlso 4% stacking gel was constituted using 30% reservoir.0.650 Acrylamide/bisacrylamide, 0.650 1.0MTris pH 6.8, 3.645 distilled water, 50µl 10% SDS, The wells were marked 1-6, with different25µl 10% APS and 5µl TEMED. The stacking gel sample added as; Maker sample, uninducedwas introduced into gel sandwich until solution sample unpurified protein sample, flow throughreached the top of front plate. sample, wash sample, and Eluted sample. 20µl each of these samples mixed with 5µl of 1xThe combs later were carefully inserted and the sample buffer heated at 100˚c for 5 minutesgel allowed to polymerize. After which the and added to the wells.3: RESULTS.3.1 Expression and protein extraction from WK6 E.coliFollowing expression usind IPTG and protein extraction through osmotic shock, the concentration of theprotein was measured using a Nanodrop™ at 280nm and found to be 119µg/µl3.2 Running Gel And staining with Commasie Blue on SDS-PAGEThe gel was run and then transferred to a small container, containing 20ml commasie blue. The gel wasdistained later using commasie distainer, after the bands were visible. Gels were preserved formolecular weight determination. MK UI UP FT WS ET 15kDa 10kDaFigure 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 membraneusing 35ml transfer buffer.
  • 6. The membrane was transferred to a smaller container, with 8ml 3% PBST buffer. And membraneblocked using 15ml 1% milk solution to prevent unspecific binding.Antibodies were poured into the solution of buffer containing the membrane, the membrane washedwith TBS 3 times, and second Antibody which had a HRP conjugate was added and washed again beforeadding the developing reagent for HRP and observed for 30minute. The membrane was washed , driedand scanned. MK UI UP FT WS ET 15kDa 10kDaFigure 2, The results of Western blotting after immune-blotting. MK is the marker, UI, uninduced sample, UP, unpurifiedprotein, 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 migrationin a gel with that of the standards. A plot of log of the molecular weight (in kDa) of each band ofstandard (Y) was done against relative distance traveled from the well (X). A line of best fit wasdrawn 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 ofMigration 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 + 2.3727Y=1.135884. Protein molecular weight was obtained by calculating the antilog of 1.135884.Protein Molecular weight =13.67kDa4: DISCUSSION.SDS-PAGE is a technique widely used in molecular biology, biochemistry, forensics, and genetics toseparate proteins according to their electrophoretic mobility, function of length of polypeptide chain ormolecular 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 weightof unknown protein was calculated as shown above indicating that the molecular weight of the proteinis 13.67 kDa .The calculation support with more confidence the prediction of the protein size .SDS PAGEresult shows that the protein was not well purified due to presence of extra small bands together withthe 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 anddetermination of the molecular size of unknown protein sample using the empirical relationshipobserved between log of molecular weight (kDa) and relative mobility on SDS-PAGEWestern 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 sizemarker or ladder in kDa, and also on protein expression. In our experiment we used Western blottechnique to confirm expression in WK6 E. coli. The Nanobody expressed contained 6x histidine tagsattached 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 whichindicated there were no protein expressed. After induction with IPTG, we had a small band on UP laneindicating that, there was protein expression. On the ET lane, a heavy band of our protein of interestwas detected and the size, determined using SDS-PAGE was approximately 13.67kDa).The sensitivity ofthe 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 demonstrateexpression of Nanobody containing 6xhistine tail and approximately estimate visually the size bycomparing with the ladder and by mathematical comparison of unknown protein distance of migrationin 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 technicalguidance and support during practical training. Not forgetting all the IPMB lecturers who provided uswith the necessary information on Molecular biology.7: REFERENCES.Amann E, Brosius J, Ptashne M. 1983. Vectors bearing a hybrid trp-lac promoter useful for regulatedexpression of cloned genes in Escherichia coli. Gene 25: 167-178Arnau J, Lauritzen C, Petersen GE, Pedersen, J.(2006) Current strategies for the use of affinity tags andtag removal for the purification of recombinant proteins. Protein Expr Purif.; 48 (1):1–13.Cohen, S.; Chang, A.; Boyer, H.; Helling, R. (1973). "Construction of biologically functional bacterialplasmids in vitro". Proceedings of the National Academy of Sciences of the United States of America 70(11): 3240–3244Esposito D, Chatterjee DK.(2006) Enhancement of soluble protein expression through the use of fusiontags. Curr Opin Biotechnol. ;17(4):353–8.Hamers-Casterman, T. Atarhouch, S. Muyldermans, G. Robinson, C. Hamers, E.B. Songa, N. Bendahmanand R. Hamers, Naturally occurring antibodies devoid of light chains. Nature, 363 (1993), pp. 446–448Kobayashi I. (2001). Behavior of restriction–modification systems as selfish mobile elements and theirimpact on genome evolution Res. 29 (18): 3742–56Nelson, PN; Reynolds, GM; Waldron, EE; Ward, E; Giannopoulos, K; Murray, PG (2000). "DemystifiedMonoclonal antibodies". Molecular pathology : MP 53 (3): 111–7.Muyldermans, Single domain camel antibodies: current status. J Biotechnol, 74 (2001), pp. 277–302Rahbarizadeh F., M.J. Rasaee, M. Forouzandeh Moghadam, A.A. Allameh, and E. Sadroddiny(2004).Hybridoma and Hybridomics. , 23(3): 151-159.Roberts RJ; Murray, Kenneth (1976). Restriction endonucleases. CRC Crit. Rev. Biochem. 4 (2): 123–64Odongo, S. IPMB General Practical Course Manual