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Construction and Evaluation of NovelFusion Proteins for Targeted Delivery ofMicro Particles to Cellulose Surfaces
William Lewis,1 Eli Keshavarz-Moore,1 John Windust,2 Donna Bushell,2 Neil Parry3
1Department of Biochemical Engineering, University College London,Torrington Place, London, WC1E 7JE, United Kingdom; telephone: 044 207 6792961;fax: 044 207 2090703; e-mail: [email protected] Research and Development, Colworth, United Kingdom3Unilever Research and Development, Port Sunlight, United Kingdom
Received 8 June 2005; accepted 19 December 2005
Published online 3 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20849
Abstract: The use of IgG antibodies and fragments hasbeen limited to specific sectors of the biotechnologyindustry due to thehigh cost of producing largebatchesofproduct necessary for alternative applications. A novelclass of Camelid antibodies, known as VHH offer a moreeconomical opportunity to meet a wider application inindustry. In this study, we report the evaluation of fourllama VHH-cellulose binding domain fusion proteinsdisplaying varying formats of VHH and CBD domains.Proteins were characterized in a targeted particle deliverysystem as amethod of delivering agents such as perfumeto laundry in the wash cycle. Fusion proteins were shownto be stable at high pH and in the presence of a detergentbase. Theywere also shown to bind effectively to both thedesignated antigen, the azo-dye reactive-red 6 (eitherconjugated to BSA or attached to coacervate microparti-cles), and cellulose. Binding strength differences wereobserved between the different fusion protein formatsusing surface plasmon resonance. The effect of keylaundry ingredients was also studied. Combining thefusion proteins and particles into a delivery and deposi-tion study generated clear microscopy evidence forbifunctionality. Confirmation of this was validated byGC-MS analysis of retained fragrance. This research,reporting the construction and characterization of avariety of fusion proteins, illustrates that the singlemultidomain fusionprotein route offers a new technologyfor successful targeted delivery of encapsulated benefitagents. Furthermore, the potential to modify or select forproteins to recognize a wide range of surfaces is alsopossible. � 2006 Wiley Periodicals, Inc.
Keywords: CBD; fusion protein; multi-domain; particle;VHH
INTRODUCTION
High development costs of antibodies (in particular during
the early stages of development) have prohibited their
broader application in high volume low value biotechnology
markets such as the home and personal care industry. Their
use has been restricted to applications that require small
quantities of antibody such as for diagnostic and therapeutic
applications where the higher cost per unit mass is acceptable
(Ghahroudi et al., 1997).
A relatively new class of antibody (Hamers-Casterman
et al., 1993) offers the potential to meet the economic
requirements for use within the wider biotechnology
industry. These antibodies originate fromCamelidae (llamas,
camels, and alpacas) and are devoid of light chains. The
antigen binding region consists of the variable domain from
the heavy chain and is described asVHH (van derLinden et al.,
2000).
The affinity and specificity of these VHH chains is
comparable to those of conventional antibody fragments
selected for the same antigens, but unlike their monoclonal
counterparts they can be upscaled at good yields in yeasts
such as Saccharomyces cerevisiae (van der Linden, 1999).
They are also reported to be considerably more temperature
stable, some showing functionality at temperatures up to
908C (van der Linden et al., 1999). It has also been reported
that the smaller size of the VHH chain reduces antigenicity
and allows binding into novel epitopes such as the active sites
of enzymes (Conrath et al., 2001; Transue et al., 1998).
Potential applications for low cost antibody fragments in the
biotechnology industry include environmental monitoring
(Churchill et al., 2002), removal of pollutants from the
environment and waste-water (Harris, 1999; Molloy et al.,
1995), detection and neutralization of microbial contami-
nants in foodstuffs and (Ercole et al., 2003; Ledeboer et al.,
2002) and catalysts in chemical reactions (Hilhorst, 1993).
They also offer the possibility for targeted delivery of agents
in products such as anti-dandruff shampoos and targeted
bleach/perfume in laundry powders and liquids (van der
Linden, 1999).
A driving force for the development of the technology into
commercially viable formats has been the production of
multi-domain proteins, containing one or more affinity
domains. Polysaccharide binding domains (PBD) have been
a protein domain of choice to combine with the antibodies as
�2006 Wiley Periodicals, Inc.
Correspondence to: E. Keshavarz-Moore
these domains offer the potential to target polysaccharide
based surfaces. The cellulose binding domain (CBD) was
selected from the PBD class as this has successfully been
shown in the past to bind to cellulosic surfaces of commercial
relevance (Davis and Parry, 2001; Perry and Clarkson, 1998).
Cellulose binding domains (CBDs) are structurally and
functionally independent, noncatalytic modules found in
many polysaccharide degrading enzymes such as cellulases
and hemicellulases (Linder and Teeri, 1997). The CBD is
connected to the rest of the cellulase enzyme by linking
segments of peptide of sufficient length and flexibility to
allow the efficient orientation and operation of the catalytic
site (Carrard et al., 2000). The main commercial application
to date of these domains is the use of CBDs in fusion proteins
as tags for affinity purification or immobilization (Linder
et al., 1998).
This study reports the combination of llama antibody
fragments with CBD in order to examine the feasibility of
using multi-domain proteins as self assembling delivery
vehicles to deliver encapsulated actives to cellulosic
surfaces. To this event an antibody fragment with a known
binding characteristic towards a dye moiety was chosen.
VHH molecules selected against the azo-dye reactive red 6
(RR6) and their production in Saccharomyces cerevisiae
have been described previously (Frenken et al., 2000; van der
Linden et al., 1999). This study describes the formation and
production of a llama anti-RR6 VHH fusion molecule bound
to a fungal CBD and the effects of the fused components on
the VHH affinity. The effects of some common components
industrial formulations on VHH affinity are also investigated
on VHH affinity to anticipate potential applications.
MATERIALS AND METHODS
All materials were obtained from Sigma-Aldrich Company
Ltd., Poole, England unless otherwise stated.
Construction of Anti-RR6-CBD Fusion Proteins
Fusion proteins were constructed, generated, and purified
according to appropriate modifications of methods described
in relevant literature using standard laboratory protocols
(Frenken et al., 2000; Harmsen et al., 2000; Nyyssonen et al.,
1993; Spinelli et al., 2001; Thomassen et al., 2002; van der
Linden et al., 1999, 2000; Verhoeyen et al., 1995; Woolven
et al., 1999, and www.microbialcellfactories.com/content/2/
1/1/).
Four proteins were produced in yeast for study: VHH anti-
RR6, VHH anti-RR6-CBD, VHH anti-RR6-VHH anti RR6-
CBD, and CBD-VHH anti-RR6-CBD. Further details of the
construction of the proteins can be found below and in
published patent WO0146357 (Davis and Parry, 2001).
Proteins were purified using a protein A based method
previously described by van der Linden et al. (1999).
A clone of the anti-RR6 protein within the vector pPIC9
was selected for use. The CBD from Trichoderma reesei,
which has known binding affinity to cellulosic substrates was
chosen. Cloning plasmid pUC19 containing VHH anti-RR6
was digested with SacI and BstEII and the fragment cloned in
a S-B digested pPIC9 already containing the CBDtr.
ACBD-VHH-CBD construct was created by using the VHH
RR6-CBD vector construct created above was as a template
to PCR—the CBD using primers 50CBD to introduce a Xho1
restriction site and 30linker to introduce a PstI restriction site.RR6 VHH-CBD was excised from the vector using the
restriction enzymes XhoI and EcoRI and then ligated into a
similarly digested vector pUC19 (Amersham Biosciences,
Little Chalfont, UK). This construct was digested with XhoI
and PstI together with the CBDPCR fragment. All fragments
were ligated in a three-step ligation to create CBD-VHHRR6-
CBD plasmid. The construct was excised from this vector
using XhoI and EcoRI and ligated into the yeast expression
vector pPIC9. The plasmid map for the CBD-VHHRR6-CBD
is shown in Figure 1.
In order to create the VHH RR6-VHH RR6-CBD the VHH
RR6-CBD vector was restriction enzyme digested using the
enzyme BstEII in order to linearize the vector. The linearized
vector containing RR6 VHH-CBD and the RR6-VHH
restricted PCR fragment were then ligated into the vector
pPIC9 according to standard molecular biology techniques.
Protein sequence data for theCBD-VHHRR6-CBD is given
as an example in Figure 2. There was no linker present
between the two VHHs in the VHH RR6-VHH RR6-CBD
protein.
Preparation of BSA-RR6 Conjugate
A 1 g/L RR6 (ICI chemicals, London, UK) solution (10 mL)
was made in pH 8.5 borate buffer (0.1 M Na2B4O7 � 10H2O,
0.05 M NaCl). Ten milliliters of a 0.1 g/L BSA solution was
also made in borate buffer. The two solutions were combined
and placed on a rotary mixer overnight at 258C. The solution
Figure 1. Plasmid map of CBD-VHHRR6-CBD in pPIC9.
626 Biotechnology and Bioengineering, Vol. 94, No. 4, July 5, 2006
DOI 10.1002/bit
was then washed with TRIS to block unused binding sites
(100mM, pH8 inmilliQwater) until the colour ran clear. The
solution was then exchanged into PBS (0.24 g/L NaH2-
PO4 �H2O, 0.49 g/L Na2HPO4 and 4.25 g/L NaCl in milliQ
water) using an Amicon stirred cell concentrator system
(10 kDa Omega membrane, Pall corporation, USA) to
remove excess dye and stored at 48C.
Analysis of RR6 Activity in Clones
High binder microtiter plates (Greiner Bio-one Ltd., Stone-
house, UK) were sensitized with 10 mg/L BSA-RR6 in PBS
or PBS only as a control overnight at 48C. Plates were thenwashed and blockedwith 1%BSA in PBST (PBSwith 0.15%
Tween-20) for 1 h at 378C. The blocking buffer was removed
and replaced with 50 mL/well yeast supernatant and 50 mLblocking buffer. A sample containing blocking buffer and
fermentationmedia (as above)was used as a negative control.
Each plate was then incubated for 1 h at 378C.Supernatants were removed and thewells washed 10 times
with PBSTusing a platewasher. Rabbit anti-llama polyclonal
sera in blocking buffer (100 mL/well, Unilever, Vlaardingen,The Netherlands) was added to each well and incubated for
1 h at 378C. The wells were washed as previously and goat
anti-rabbit alkaline phosphatase conjugate (100 mL/well,Zymed Laboratories, Inc., San Francisco) added at 1/1,000 v/
v dilution in blocking buffer. Samples were then incubated
for 1 h at 378C and washed as previously. Alkaline phos-
phatase substrate para-NitroPhenylPhosphate (pNPP) (1 g/L)
in a pH 9.8 substrate buffer (105.1 g/L Di-ethanolamine,
20.3 g/L MgCl2 � 6H2O in milliQ water) was added to each
well in 100 mL aliquots. After the color had developed the
absorbance at a wavelength of 405 nm was measured.
Analysis of CBD Activity of Clones
Ethyl cellulose (20 mL of a 1% w/v suspension) and Marvel
(milk whey, 80 mL of 0.1% w/v suspension) in PBST was
added to wells of a 0.45 mm PTFE membrane filter plate
(Millipore (UK) Ltd., Watford, England) and incubated on a
shaker at room temperature for 1 h. The blocking buffer was
removed with a vacuum and 50 mL yeast supernatant and
50 mL blocking buffer was added to the wells. A sample
containing only blocking buffer and media was used as a
negative control. Incubation was on a shaker for 1 h at room
temperature. The supernatants were removed and the wells
washed 10 times with PBST.
Rabbit anti-llama polyclonal sera (100 mL/well, as above)were added to eachwell at an appropriate dilution in blocking
buffer and incubated as previously. Thewells werewashed as
before and 100 mL of goat anti-rabbit alkaline phosphatase
conjugate (identical conditions as above). Samples were
incubated and washed as before. Substrate (pNPP/DEA, as
above) was added and the color allowed to develop prior to
absorbance measurement at 405 nm.
Stability of anti-RR6 VHH-CBD
Samples of anti-RR6 VHH-CBD (200 mL of a 1 g/L solution)
were incubated in 0.1MHEPES (pH 8), 0.1MCHES (pH 9),
CAPS (pH 10) and OMO base (2 g/L, Unilever Vlaardingen,
as above) in milliQ ultrapure water for 0 min (fusion added,
mixed and removed immediately), 30 and 90 min. Samples
were then analyzed by SDS–PAGE using 12.5% Tris-Cl
Ready gels (Bio-Rad, Hemel Hampstead, England).
Determination of Binding Affinities of FusionMolecules Using the Biacore 2000
The RR6-BSA conjugate (prepared as previously described)
was immobilized on a CM5 (Carboxymethyl Dextran
surface, Biacore AB., Stevenage, England) using a Biacore
2000 and standard BIAcore EDC/NHS binding procedures.
One flow cell was kept as a blank control, and one other flow
cell coated with 5000 RU of RR6-BSA conjugate.
TBS-Twas used as a running buffer (12.1 g/LTrizma base,
8.7 g/L NaCl and 0.05 mL/L Tween-20 in milliQ water,
pH 7.4), and the chip was regenerated with 50 mMHCl after
each use. The analyzedmolecules were diluted to between 40
and 200 nM in the running buffer and passed through the cell
at 30 mL/min for 60 s before being washed in 0.76 mg/L RR6
in running buffer.
Fusion protein molecules and their ability to bind to the
RR6 chip were also analyzed in the presence of common
laundry detergent components (alkaline silicate, sodium
carbonate, sodium sulfate, and sodium tri poly phospate
(STPP)) at concentrations used in commercial brands.
Deposition of Particles on CelluloseFragments and Cotton Yarn
Coacervate particles containing sunflower oil and RR6 were
prepared according to protocols derived from conditions
in the available literature (Tolstoguzov, 1997). Positively
charged latex particles containing perfume were kindly
provided by Unilever R&D group at Port Sunlight. RR6
coated coacervate particles (1 ml of a 200 g/L suspension)
were incubated in a 10 g/L solution of Sigma cell with and
without 1 mg/mL anti-RR6 VHH-CBD for 60 min and a
sample studied under a microscope for aggregation as
anticipated by molecular binding of the fusion protein
domains.
Figure 2. Sequence of the CBD-VHHRR6-CBD.
Lewis et al.: Fusion Proteins in Targeted Particle Delivery 627
Biotechnology and Bioengineering. DOI 10.1002/bit
The anti-RR6 VHH-CBD fusion protein was incubated
with the particles for 60min, thenwashedwith PBS for 15min
at 258C. Pre-washed cotton strands were added to the fusionprotein/particle mixture and mixed for 60 min under
designated washing conditions. Strands were then rinsed
after this period andmounted formicroscopy or processed for
volatile organic compound analysis by GCMS.
Analysis of Volatile Organic Components ofLatex Particles Deposited on Cotton
Following incubation, cotton strands were mixed with ethyl
acetate for 30min at 258C. Samples were sonicated for 2min
using a sonicating water bath and the ethyl acetate removed
and analyzed by GCMS for volatile organic components.
RESULTS AND DISCUSSION
Demonstration of RequiredFunctionality of Fusion Proteins
Production and purification of the proteins was done on a
laboratory scale, with the main focus being on procuring a
small amount of the necessary protein in order to perform
characterization studies.
The selection of the antiRR6-CBD is shown to illustrate
the methodology, and all other proteins were selected using
the same procedure. Data showing the binding of crude
supernatants of yeast clones containing antiRR6-CBD fusion
protein to RR6 is shown in Figure 3. PBS is included as a
negative control. A similar level of binding to RR6 can be
seen in all clones. This indicates a level of antibody domain
functionality from all clones and suggests that the domain has
folded correctly during fermentation in all samples.
The binding of each clone to ethyl cellulose particles was
also assayed to insure activity of the CBD and thereby
confirm bifunctionality (Fig. 4). The media used in the
fermentation was also tested for ethyl cellulose particle
interaction as a negative control. Notably clones 4 and 5
illustrated no binding to cellulose. This was then correlated
with a non-expression status of these clones as determined by
SDS–PAGE that showed that the supernatant of clones 4 and
5 contained only theVHH domain and therefore not expressed
theCBDdomain (data not shown).All other clones examined
showed specific binding for ethyl cellulose thus indicating
that the cloning of the CBD fragment was successful.
Binding of VHHRR6-VHHRR6-CBD, CBD-VHHRR6-CBD
fusion proteins were similarly analyzed and the procedure
used to select suitable clones, where notably the fragment
with two CBDs demonstrated enhanced deposition to
cellulose (data not shown).
Prior to testing the fusion protein for dual activity it was
important to test its stability in pH conditions similar to those
of laundry detergents. Figure 5 illustrates that the protein
was not degraded into individual components even after
90 min in a washing powder base formulation (OMO base).
The laundry detergent OMO is a commercially available
European detergent that operates at a high pH and contains
many chemical components that, in combination with the pH
may degrade the protein. The protein was also shown to be
stable at pH 8–10 for 90 min.
Figure 3. Binding of VHH-CBD fusion proteins produced by clones 1 to 8
to RR6-BSA and PBS as determined in indirect ELISA.
Figure 4. Binding of VHHRR6-CBD fusion proteins produced by clones
1–8 to ethyl cellulose as determined by indirect ELISA. Fermentationmedia
is included as a negative control.
Figure 5. Effect of time, pH, and detergest base on the stability of
VHHRR6-CBD protein as demonstrated by incubation for 0, 30, and 90
minutes at pH 8, 9, 10 and in the presence ofOMObase and subsequent SDS-
PAGE. The arrow demonstrates the position of the fusal protein.
628 Biotechnology and Bioengineering, Vol. 94, No. 4, July 5, 2006
DOI 10.1002/bit
Determination of RR6 Binding Strengths
Figure 6 illustrates the binding affinities of the different
fusion molecules as determined by surface plasmon reso-
nance. All results were calculated with BIAcore’s BIAeva-
luation software. Association/dissociation constants were
calculated independently with the software using a 1:1
Langmuir isotherm. All curves used in calculations were
subtracted from those obtained from the blank flow cell to
remove background effects.
Published affinities for IgG antibodies are in the nMol
(0.5–350) (Beresford et al., 1999; Jansson et al., 1997; Quinn
and O’Kennedy, 2001). It is this range that is deemed
necessary to show that the protein has the required func-
tionality towards the antigen. VHH antibody affinities are
commonly slightly lower than corresponding IgG’s (van der
Linden, 1999), however, other advantages as described above
more than compensate.
Data in Figure 6 indicates that the single VHH fragment
possesses the highest binding affinity of approximately 4.5 nM,
and the addition of a CBD reduced the binding affinity to
23 nM. However, this drop in affinity appears to be
counteracted by the addition of a second VHH domain in
the fusion protein as this VHH-VHH-CBD demonstrated an
affinity of about 10 nM. The CBD-VHH-CBD molecule
format shows an affinity of about 20 nM, indicating that
adding a further CBD did not have a significant negative
effect.
The box section of the graphs shows the area in which 75%
of the data points lie and is taken as the range of affinities the
protein exhibits. In some cases this range appears to be quite
large, this is potentially due to the presence of the detergent
components affecting the reaction and/or the Biacore sensor.
Error bars show the 5th/95th percentiles, and once again vary
between sample, possibly due to interference from laundry
components.
It can be seen that the proteins conform to the desired range
of activity as described above. Spinelli et al., 2004 have
previously published affinities for a range of single anti-RR6
VHHmolecules between 22 and 83 nMas determined by Iasys
biosensor. This difference could be due to a difference in
protein structure and consequently function or the difference
in method of affinity determination used.
It is postulated that the reduction in affinity caused by the
presence of the CBD is caused by a greater steric hindrance
due to the increased size of the molecule. This effect was
apparently limited, as the addition of another CBD had no
greater detrimental effect than that of the single CBD. This
may suggest that optimizing themolecular structure between
the CBD and the first antibody fragment may return the
antibody binding strength back to its single domain counter-
part. The addition of another VHH, as indicated in the
VHH-VHH-CBD protein, reduced this hindrance by offering
another binding site.
Figure 7a–c shows the effect of different detergent
components on the fusion protein affinities. Binding affinities
were calculated as previously described. The effect of the
components plotted in Figure 7a–c appears to vary with
fusion molecule. The most stable of the fusion proteins
appeared to be the VHH-RR6-VHHRR6-CBD that maintained
an affinity of about 10 nM in the presence of each component.
STPP, which significantly reduced the affinity of the single
VHH molecule by about 35 nM, appeared to have little or no
effect on the other fusion molecules. It is plausible that
the presence of the CBD conveys an extra stability to the
molecule, perhaps by affecting the charge layer around the
molecule, or by physically preventing the STPP from
interacting with the VHH component. The CBD-VHH-CBD
molecule shows a similar trend to that of theVHH-CBD and is
not displayed here.
Figure 7d shows the affinities of the various fusions in the
presence and absence of alkaline silicate. It can be seen that
alkaline silicate has a detrimental affect on the fusion
molecules to a large extent, in particular with the single VHH
molecule, reducing the affinity to approximately 550 nM.
TheVHH-CBDandVHH-VHH-CBDaffinitieswere reduced to
approximately 250 nM, and the CBD-VHH-CBD to approxi-
mately 300 nM. This appears to suggest that the presence of
the CBD, whilst having a minor negative effect on antibody
affinity in an idealized condition, convey an additional
stability in more extreme reaction conditions.
Determination of Bifunctionality of Fusion ProteinVHHRR6-CBD-Binding to Cellulose and RR6
Preliminary tests of binding ability of particles to the surface
of cellulose in the presenc of fusion were carried out using
cellulose fragments (Sigmacell). Figure 8 illustrates a
microscopy test utilizing RR6 coated coacervate particles
and Sigmacell cellulose in the presence and absence of RR6
VHH-CBD fusion. It can be seen that powdered Sigmacell
cellulose is bound to the coacervate particle only in the
presence of the fusion protein indicating the dual CBD and
antibody activities resulting in cross-linking.
An extension of the approach using cotton fabric instead of
Sigmacell is shown in Figure 9. Particles firstly incubated
Figure 6. Binding affinities of VHHRR6, VHHRR6-CBD,
VHHRR6VHHRR6-CBD, and CBD-VHHRR6-CBD as determined by surface
plasmon resonance. Middle lines represent the median, the boxes represent
the interquartile range, and the whiskers represent the 5th/95th percentiles.
Lewis et al.: Fusion Proteins in Targeted Particle Delivery 629
Biotechnology and Bioengineering. DOI 10.1002/bit
with fusion and then further mixed with a cotton strand were
comparedwith particles alone incubatedwith a cotton strand.
It is clear that enhanced binding of particles occurred in
the presence of the fusion protein. An increase in particle
deposition is necessary to prove the efficacy of the protein in
this system, and shows the protein could be used one of two
ways: to increase deposition of available particles (and
convey increased benefit) or give comparable depositionwith
reduced amount of particles (and convey reduced costs).
Although Figure 9 shows a good visual benefit, there is a
need for some quantifiable data regarding the deposition of
the particles. As such a GCMS based method to analyze
volatile organic compounds in RR6 coated latex spheres was
used. This permitted the volatile components to be quantified
in the presence and absence of the fusion protein, as
demonstrated in Figure 10. The volatile components can be
seen to be in substantially higher concentrations in the
presence of fusion molecule, thereby confirming that the
fusion has the desired effect of increasing the perfume
deposition to cotton.
CONCLUSIONS
Fusion protein molecules containing VHH anti-dye domains
and cellulose binding domains in various formats were
created and successfully expressed in yeast. Individual
Figure 7. a–c: Binding affinities of protein in the presence of laundry detergent components as determined by surface plasmon resonance. Middle lines
represent the median, the boxes the interquartile range and the whiskers the 5th/95th percentiles. C: Control; STPP: Sodium Tri-Poly-Phosphate; SN: Sodium
Nitrate; SC: Sodium Carbonate. a: VHHRR6; b: VHHRR6-CBD; c: VHHRR6-VHHRR6-CBD. d: Binding affinities of the different fusions in the presence and
absence of alkaline silicate. C: Control (only fusion); S: alkaline silicate; V: VHHRR6; VC: VHHRR6-CBD; VVC: VHHRR6-VHHRR6-CBD; CVC:
CBDVHHRR6-CBD. The middle line represents the median of the data, the boxes the interquartile range and the whiskers the 5th/9th percentiles.
Figure 8. a: Control—coavervate particle incubated with Sigmacell. b: Test—coacervate particle incubated with Sigmacell in the presence of fusion protein
VHHRR6-CBD. Sigmacell can be seen to be adhering to the coacervate particle in the presence of the fusion protein.
630 Biotechnology and Bioengineering, Vol. 94, No. 4, July 5, 2006
DOI 10.1002/bit
clones producing protein of the anticipated molecular weight
for each CBD fusion construct demonstrated binding activity
to both RR6 and cellulose. The best clones were further
selected for production in 3L scale fermentations to generate
material for further studies.
The binding affinity of the fusion molecules was
determined indicating that under idealized conditions
(TBS-T buffer) the single VHH fragment possessed the best
antibody binding affinity. Although CBD addition to the
molecule reduced the antibody affinity in an idealized
condition, in the presence of some laundry components it
appeared to convey an added stability. Themost stable fusion
molecule in the presence of laundry agents was the VHH-
VHH-CBD format. Here, the addition of the extra VHH also
served to partly counteract the observed loss of affinity
identified with the addition of a CBD.
The VHH-CBD molecule demonstrated a cross linking
capability between cellulose and RR6 sensitised coacervate
particles indicating a bifunctional fusion protein had been
successfully generated. When extended to experiments with
perfume encapsulates it was further demonstrated that the
fusion protein was capable of improving the deposition of
fragrance. Therefore, we can conclude that the approach of
using fusion proteins for targeted active delivery has
significant potential not only in the laundry area but also in
the deposition of actives to other commercially important
surfaces. A significant increase in deposited particles shows
that further investigation of this system is warranted.
Future work will be focused on developing antibody
binding domains that bind to the capsule wall material itself
and optimizing this protein domain with a number of CBD
molecule options. Further characterization will determine
efficiency of the delivery system under different conditions
and optimize levels of protein used.
NOMENCLATURE
CBD cellulose binding domain
PBS phosphate buffered saline
TBS-T tris buffered saline with tween-20
GC-MS gas chromatography mass spectrometry
VHH variable heavy chain fragment from heavy chain llama antibodies
PBD polysaccharide binding domain
GCMS gas chromatography mass spectrometry
RR6 azo dye reactive red 6
OMO commercially available detergent
References
Beresford GW, Pavlinkova G, Booth BJM, Batra SK, Colcher D. 1999.
Binding characteristics and tumor targetting of a covalently linked
divalent CC49 single chain antibody. Exp Cancer 6:911–917.
Figure 9. a and b: Control—cotton strands incubatedwith coacervate particles (a, 10000�magnification; b, 40000�magnification). c and d: Test—Control
incubated with coacervate particles in the presence of fusion protein VHHRR6-CBD (c, 10000� magnification; d, 40000� magnification).
Figure 10. Volatile organic compounds listed in order of descending
volatility, in the presence and absence of fusion protein VHHRR6-CBD.
Lewis et al.: Fusion Proteins in Targeted Particle Delivery 631
Biotechnology and Bioengineering. DOI 10.1002/bit
Carrard G, Koivula A, Soderlund H, Beguin P. 2000. Cellulose-binding
domains promote hydrolysis of different sites on crystalline cellulose.
Proc Natl Acad Sci USA 97:10342–10347.
Churchill RLT, Sheedy C, Yau KYF, Hall JC. 2002. Evolution of antibodies
for environmental monitoring: From mice to plants. Anal Chim Acta
468:185–197.
Conrath KE, LauwereysM, GalleniM,MatagneA, Frere JM, Kinne J,Wyns
L,Muyldermans S. 2001. Beta-lactamase inhibitors derived from single
chain antibody fragments elicited in the Camelidae. Antimicrob Agents
Chemother 45:2807–2812.
Davis PJ, ParryNJ. 2001. Detergent composition comprising benefits. Patent
no. WO0146357.
Ercole C, Del Gallo M, Mosiello L, Bacella S, Lepidi A. 2003. Escherichia
coli detection in vegetable food by a potentiometric biosensor. Sensor
Actuat B-Chem 91:163–168.
Frenken LGJ, van der Linden RHJ, Hermans PWJJ, Bos JW, Ruuls RC, de
Geus B, Verrips CT. 2000. Isolation of antigen specific llama VHH
antibody fragments and their high level secretion by Saccharomyces
cerevisiae. J Biotechnol 78:11–21.
Ghahroudi MA, Desmyter A, Wyns L, Hamers R, Muyldermans S. 1997.
Selection and identification of single domain antibody fragments from
camel heavy chain antibodies. FEBS Lett 414:521–526.
Hamers-castermanC,AtarhouchT,Muyldermans S, RobinsonG,HamersC,
Songa EB, Bendahman N, Hamers R. 1993. Nature 363:446–448.
HarmsenMM, Ruuls RC, Nijman IJ, Niewold TA, Frenken LGJ, De Geus B.
2000. Llama heavy-chain V regions consist of at least four distinct sub-
families revealing novel sequence features. Mol Immunol 37:579–590.
Harris B. 1999. Exploiting antibody based technologies to manage
environmental pollution. Trends Biotechnol 17:290–296.
Hilhorst R. 1993. Catalytic antibodies—newdevelopments. Anal ChimActa
279:129–134.
JanssonM,HallenD,KohoH,AnderssonG, Berghard L, Heidrich J, Nyberg
E, UhlenM, Kordel J, Nilsson B. Characterization of ligand binding of a
soluble human insulin-like growth factor I receptor variant suggests a
ligand-induced conformational change. J Biol Chem 28: 8189–8197.
Ledeboer AM, Bezemer S, de Haard JJW, Schaffers IM, Verrips CT, van
Vliet C, Dusterhoft EM, Zoon P, Moineau S and Frenken LGJ. 2002.
Preventing phage lysis of Lactococcus lactis in cheese production using
a neutralizing heavy chain antibody fragment from llama. J Dairy Sci
85:1376–1382.
Linder M, Teeri TT. 1997. The roles and functions of cellulose binding
domains. J Biotechnol 57:15–28.
Linder M, Nevanen T, Soderholm L, Bengs O, Teeri TT. 1998. Improved
immobilization of fusion proteins via cellulose binding domains.
Biotechnol Bioeng 60:642–647.
Molloy P, Brydon L, Porter AJ, Harris WJ. 1995. Separation and
concentration of bacteria with immobilized antibody fragments. J Appl
Bacteriol 78:359–365.
NyyssonenE, PenttilaM,Harkki A, SaloheimoA,Knowles JKC,Keranen S.
1993. Efficient production of antibody fragments by the filamentous
fungus Trichoderma reesei. Biotechnology (N Y) 11:591–595.
PerryA, Clarkson JC. 1998.Detergent composition. Patent no.WO9800500.
Quinn JG, O’Kennedy R. 2001. Biosensor-based estimation of kinetic and
equilibrium constants. Anal Biochem 290:36–46.
Spinelli S, Tegoni M, Frenken L, van Vliet C, Cambillau C. 2001. Lateral
recognition of a dye hapten by a llama VHH domain. J Mol Biol 311:
123–129.
Spinelli S, Desmyter A, Frenken L, Verrips T, TegoniM, Cambillau C. 2004.
Domain swapping of a llama VHH domain builds a crystal-wide beta-
sheet structure. FEBS Lett 564:35–40.
Thomassen YE, Meijer W, Sierkstra L, Verrips CT. 2002. Large-scale
production of VHH antibody fragments by Saccharomyces cerevisiae.
Enzyme Microb Technol 30:273–278.
Tolstoguzov VB. 1997. Protein polysaccharide interactions. In: Food
proteins and their applications. New York: Marcel Dekker, Inc.
Transue TR, de Genst E, Gharoudi MA, Wyns L, Muyldermans S. 1998.
Camel single domain antibody inhibits enzyme my mimicking
carbohydrate substrate. Proteins 32:515–522.
van der Linden R. 1999. Unique characteristics of llama heavy chain
antibodies. PhD thesis, Utrecht University.
van der Linden RH, Frenken LGJ, de Geus B, HarmsenMM, Ruuls RC, Stok
W, de Ron L, Wilson S, Davis P, Verrips CT. 1999. Comparison of
physical chemical properties of llama VHH antibody fragments and
mouse monoclonal antibodies. Biochim Biophys Acta 1431:37–46.
van der Linden R, de Geus B, Stok W, Bos W, van Wassenaar D, Verrips T,
Frenken L. 2000. Induction of immune responses andmolecular cloning
of the heavy chain antibody repertoire of Lama Glama. J Immunol
Methods 240:185–195.
Verhoeyen ME, van der Logt CPE, Beggs TS, Davies PJ. 1995. Antibody
fragments for controlled delivery of therapeutic agents. Biochem Soc
Trans 23:1067–1073.
Woolven BP, Frenken LGJ, van der Logt P, Nicholls PJ. 1999. The structure
of the llama heavy chain constant genes reveals a mechanism for heavy-
chain antibody formation. Immunogenetics 50:98–101.
632 Biotechnology and Bioengineering, Vol. 94, No. 4, July 5, 2006
DOI 10.1002/bit
