Supplementary Information:
Synthesis and properties of crosslinked
recombinant pro-resilin
Christopher M. Elvin*, Andrew G. Carr, Mickey G. Huson, Jane M. Maxwell,
Roger D. Pearson, Tony Vuocolo, Nancy E. Liyou, Darren C.C. Wong, David
J. Merritt and Nicholas E. Dixon
* Author to whom correspondence should be addressed: CSIRO Livestock Industries,
Queensland Bioscience Precinct, St Lucia 4072, Australia.
Telephone: (+617) 32142506
FAX: (+617) 32142900
e-mail: chris.elvin@csiro.au
1.
2.
Methods
1.1
PCR of CG15920 Exon 1
1.2
CG15920 sequence (ex D. melanogaster Oregon R)
1.3
Amino acid sequence of rec1-resilin
1.4
DNA sequence analysis: strain specific differences
1.5
Expression of recombinant rec1-resilin
1.6
Purification of recombinant rec1-resilin
1.7
Peroxidase crosslinking of recombinant rec1-resilin
1.8
Photochemical crosslinking of recombinant rec1-resilin
1.9
Preparation of dityrosine standard and analysis
1.10
Analysis of dityrosine in rec1-Resilin samples
Time course of dityrosine crosslinking. Figure 1 illustrates: A. Dityrosine content; B.
SPM probe penetration and C. Resilience
3.
Scanning probe microscopy (SPM) study
4.
Tensile testing. Figures 2 & 3 illustrate both theoretical and experimental stress-strain
data.
5.
Expression profiles of the Drosophila resilin gene CG15920 including sequence of
primers used in RT-PCR experiments.
1
1.
Methods
1.1
PCR of CG15920 Exon 1
The rec1-resilin expression primers were ResF3 and RespepR1. Their sequences are: ResF3:
5'…CCCATATGCACCATCACCATCACCATCCGGAGCCACCAGTTAACTCGTATCTACC…3'
and RespepR1: 5'…CCGAATTCCTATCCAGAAGCTGGGGGTCCGTAGGAGTCGGAGGG…3’
These primers amplify the majority of the first exon of the CG15920 gene, encoding the
elastomeric domain, from nucleotide 48,275 to 49,183 [1]. We have named the recombinant
protein derivative encoded by this part of the CG15920 gene, rec1-resilin. Genomic DNA
was isolated from adult Drosophila melanogaster (Oregon-R) using standard techniques
(DNAzol™). Genomic DNA (450 ng) was used in a PCR reaction with the primers ResF3
and RespepR1 in a total volume of 50 µl of Advantage Klentaq buffer, containing 1 µg
primer ResF3, 1 µg primer RespepR1, 0.4 mM each dNTP, and 2 units of Advantage Klentaq
DNA polymerase. Forty cycles of amplification were carried out using the following
parameters: 1 min at 94.5 °C, 1 min at 60 °C, 1 min at 72 °C. The PCR product (946 bp) was
inserted into the pCRBlunt vector (Invitrogen) and clones were selected for resistance to
kanamycin (50 µg/ml). The exon 1 DNA fragment was prepared for insertion into the
bacterial expression vector pETMCSI [2] by partial digestion with NdeI and EcoRI. The
NdeI/EcoRI fragment was inserted between the corresponding sites of pETMCSI; the
recombinant expression plasmid, pETMCSI/Res5, was isolated from E. coli Top10 cells with
selection for ampicillin resistance and the correct construct was verified by DNA sequence
analysis (below) of the recombinant expression plasmid isolated from a single transformant
using standard procedures.
2
1.2
CG15920 sequence (ex D. melanogaster Oregon-R)
The DNA fragment used for rec-1 resilin expression is shown in bold face type.
CCGGAGCCACCAGTTAACTCGTATCTACCTCCGTCCGATAGCTATGGAGCACCGGGTCAGAG
TGGTCCCGGCGGCAGGCCGTCGGATTCCTATGGAGCTCCTGGTGGTGGAAACGGTGGACGGC
CCTCAGACAGCTATGGCGCTCCAGGCCAGGGTCAAGGACAGGGACAAGGACAAGGTGGATAT
GCAGGCAAGCCCTCAGATTCCTATGGAGCTCCTGGTGGTGGAAATGGCAACGGAGGTCGTCC
ATCGAGCAGCTATGGCGCTCCTGGCGGTGGAAACGGCGGTCGTCCTTCGGATACCTACGGTG
CTCCTGGTGGCGGAAATGGTGGACGCCCGTCGGACACTTATGGTGCTCCTGGTGGTGGTGGA
AATGGCAACGGCGGACGACCTTCAAGCAGCTATGGAGCTCCTGGTCAAGGACAAGGCAACGG
AAATGGTGGTCGCCCATCGAGCAGCTATGGCGCTCCTGGTAGTGGAAACGGCGGTCGTCCTT
CGGATACCTACGGTGCTCCTGGTGGTGGAAACGGTGGTCGTCCTTCGGACACTTACGGCGCT
CCTGGTGGCGGCAATAATGGCGGTCGTCCTTCAAGCAGCTACGGCGCTCCTGGTGGTGGAAA
CGGTGGTCGTCCATCTGACACCTATGGCGCTCCTGGTGGCGGTAACGGAAACGGCAGCGGTG
GTCGTCCCTCAAGCAGCTATGGAGCTCCTGGTCAGGGCCAAGGTGGATTTGGTGGTCGCCCA
TCGGACTCCTATGGTGCTCCTGGTCAGAACCAAAAACCATCAGATTCATATGGCGCCCCTGG
TAGCGGCAATGGCAACGGCGGACGTCCTTCGAGCAGTTATGGAGCTCCAGGCTCAGGACCTG
GTGGCCGACCCTCCGACTCCTACGGACCCCCAGCCTCTGGATCGGGAGCAGGTGGCGCTGGA
GGCAGTGGACCCGGCGGCGCTGACTACGATAACGATGAGCCCGCCAAGTACGAATTTAATTA
CCAGGTTGAGGACGCGCCCAGCGGACTCTCGTTCGGGCATTCAGAGATGCGCGACGGTGACT
TCACCACCGGCCAGTACAATGTCCTGTTGCCCGACGGAAGGAAGCAAATTGTGGAGTATGAG
GCCGATCAGCAGGGCTACCGGCCACAGATCCGCTACGAAGGTGATGCCAACGATGGCAGTGG
TCCCAGCGGTCCTGGAGGTCCTGGCGGTCAGAATTTGGGTGCCGATGGCTACTCCAGTGGAC
GTCCCGGCAATGGAAATGACAACGGAAATGGCGGTTACTCCGGTGGACGTTCAGGAGGTCAG
GATTTGGGACCTAGTGGATATTCCGGTGGTCGTCCAGGAGGTCAGGATCTAGGCGCCGGTGG
CTACTCCAATGGCAAGCCGGGCGGCCAAGACTTGGGACCAGGCGGTTACTCCGGCGGTCGCC
CTGGCGGTCAGGACTTGGGTCGAGACGGCTACTCCGGTGGACGTCCAGGTGGTCAGGACCTC
GGTGCCGGCGGCTACTCCAATGGCAGGCCAGGCGGCAATGGCAACGGTGGATCCGATGGCGG
TCGTGTGATCATCGGTGGACGTGTGATAGGCGGCCAGGATGGCGGTGATCAGGGCTACTCCG
GCGGACGTCCCGGTGGTCAGGATCTTGGACGTGATGGCTACTCCAGCGGTCGTCCTGGTGGT
CGGCCAGGCGGCAACGGCCAGGATAGTCAGGATGGCCAAGGATACTCGAGCGGCAGGCCGGG
TCAGGGTGGCCGGAATGGATTCGGACCCGGTGGTCAGAACGGTGACAACGATGGCAGCGGTT
ATCGGTACTAG
1.3
Amino acid sequence of rec1-resilin
MHHHHHHPEPPVNSYLPPSDSYGAPGQSGPGGRPSDSYGAPGGGNGGRPSDSYGAPGQGQGQ
GQGQGGYAGKPSDSYGAPGGGDGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTY
GAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGR
PSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPGQGQ
GGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGNGGRPSSSYGAPGSGPGGRPSDSYGPPASG
*
1.4
DNA sequence analysis: strain specific differences
The DNA sequence of the insert was compared to the DNA sequence of the CG15920 gene
and restriction sites including the internal NdeI site were identified. However, when the PCR
product obtained from this D. melanogaster Oregon-R strain was compared to the annotated
DNA sequence (release 3) of D. melanogaster, there were 17 apparent strain-specific
3
sequence polymorphisms (1/53 bp; shown in bold red in sequence represented in part 1.2) in
the rec1-resilin coding sequence of 909 bp. This frequency of strain-specific sequence
polymorphism was almost 3-fold higher than observed elsewhere [3]. That investigation
detected sequence polymorphisms between D. melanogaster FRT and EP strains, on average,
once every 200 bp and between each of these strains and the Release 2 genome sequence
once every 157 and 153 bp, respectively. These sequence polymorphisms resulted in three
amino acid substitutions in the coding sequence of recombinant rec1-resilin protein.
1.5
Expression of recombinant rec1-resilin
The recombinant pETMCSI/Res5 plasmid was transformed into the rne131 E. coli strain
BL21Star™(DE3)/pLysS with selection for both ampicillin (100 µg/ml) and chloramphenicol
(34 µg/ml) on LB plates containing 50 mM glucose. The strain was grown overnight at 37 °C
in similarly-supplemented LB medium, and used to inoculate a 1-litre culture containing
twice strength LB medium with ampicillin (200 µg/ml) and chloramphenicol (34 µg/ml) to
A600 = 0.05. This culture was grown to A600 = 1.0 at 37 °C with vigorous aeration in a 2-litre
baffled flask, whereupon it was treated for a further 4 h with IPTG at a final concentration of
1 mM to induce rec1-resilin production. Cells were collected by centrifugation (10,000g, 20
min at 4 °C) and the cell pellet frozen at –80 °C.
1.6
Purification of recombinant rec1-resilin
Soluble proteins were isolated following lysis and sonication of the resuspended pellet in a
buffer containing 50 mM Tris/HCl pH 7.2, 1 mM benzamidine, 1.0% Triton X-100, 10 mM
-mercaptoethanol, 10 mM imidazole and protease inhibitor cocktail (EDTA-free; Roche).
Cell disruption was carried out using an Ultrasonics™ (Melbourne, Australia) A180
sonicator (180 W maximum power output) with a 10-mm ultrasonic probe. The cell
4
suspension (40 ml) was sonicated (for 30 sec x 3) in an ice water bath. The soluble protein
fraction was collected by centrifugation at 100,000g for 1 h at 4 °C. The pH of this fraction
was readjusted to pH 7.2 with HCl. It was then applied to a Q-Sepharose column.
Recombinant rec1-resilin was washed through the column by elution with a buffer containing
50 mM Tris/HCl pH 7.2, 1 mM benzamidine, 0.1% Triton X-100, 5 mM -mercaptoethanol.
This flow-through fraction was readjusted to pH 8.2 using NaOH, then NaCl was added to
500 mM and imidazole to 10 mM. Any precipitate was removed by filtration through a 0.45µm filter. The His6 tagged rec1-resilin was affinity-purified using Ni-NTA agarose (Qiagen).
Most of the remaining contaminants passed through the Ni-NTA column in the flowthrough.
The remainder were removed with a 50 mM imidazole wash in a buffer containing 100 mM
NaH2PO4, 10 mM Tris/HCl, 500 mM NaCl, 1 mM benzamidine, 0.1% Triton X-100, 10 mM
-mercaptoethanol, pH 8.0. The recombinant protein was subsequently eluted from the resin
using a gradient of 50– 500 mM imidazole; the rec1-resilin protein eluted at ca. 80 mM
imidazole. This purification gave very pure recombinant rec1-resilin as judged by SDSPAGE analysis (see Figure 1); yields were about 15 mg per litre of culture. While the
apparent molecular weight was about 47,000 by mobility on SDS-PAGE, separate analysis
by sedimentation equilibrium measurements and capillary HPLC in SDS buffer gave values
near 23,000; the calculated value for the His6-tagged protein is 28,492. N-terminal amino
acid sequence analysis (Procise) was carried out on purified recombinant resilin. The
following sequence (at 120 pmol yield) was obtained for the first 12 amino acid residues:
MHHHHHHPEPPV, as expected from DNA sequence analysis.
1.7
Peroxidase crosslinking of recombinant rec1-resilin
Successful formation of solid polymerised rec1-resilin was obtained using Arthromyces
ramosus peroxidase to catalyse dityrosine formation. We compared the efficiency of resilin
5
crosslinking by three different peroxidases. Horseradish peroxidase (HRP), lactoperoxidase
and Arthromyces ramosus peroxidase were obtained from a commercial source (Sigma). For
crosslinking studies, we followed the procedure described by Malencik and Anderson [4] and
treated samples of soluble recombinant rec1-resilin with each of the enzymes and started the
reactions by addition of 10 mM H2O2. For the reactions described here, we used 50 µg of
each peroxidase per mg of purified rec1-resilin. The reactions were carried out for 1 h at 37
°C and samples were analysed by SDS-PAGE on 10% precast gels (Invitrogen). The results
showed that lactoperoxidase catalysed a small amount of crosslinking, while HRP led to a
significant degree of crosslinking characterised by a ladder of polymerised monomer units.
The Arthromyces peroxidase catalysed the apparently quantitative conversion of soluble
recombinant resilin into a very high molecular weight species which barely entered the 10%
SDS-PAGE separating gel.
Fluorometric analysis showed corresponding differences in the rates of dityrosine formation
catalysed by the three peroxidases. Only the Arthromyces peroxidase caused a significant
increase in fluorescence of soluble rec1-resilin. This result is consistent with the results of
Malencik and Anderson [4], who showed that the Arthromyces peroxidase was far more
effective at catalysing intermolecular dityrosine formation in calmodulin solutions than four
other peroxidases tested (HRP, lactoperoxidase, soybean peroxidase and myeloperoxidase).
1.8
Photochemical crosslinking of recombinant rec1-resilin
A photochemical method was used to crosslink the soluble rec1-resilin into a solid material.
The method described by Fancy and Kodadek [5] was used with slight modification. The
light source chosen for the present study was a 600-W tungsten-halide source (2 x 300-W
lamps; GE #38476). The spectral output showed a broad peak from 300–1200 nm. The
6
amount of [Ru(bpy)3 ]2+ was titrated using a fixed concentration (10 mM) of the oxidant
ammonium persulfate. At a protein concentration of 40 µM (1 mg/ml), about 4 µM [Ru(bpy)3
]2+ was required to achieve quantitative crosslinking to very high molecular weight
aggregates (as determined by SDS-PAGE). For production of solid samples of crosslinked
recombinant rec1-resilin, a solution of 200 mg/ml of protein was pipetted into various glass
moulds which had been pre-treated with Coatasil™ (1,1-dichloro-1fluoroethane:dimethyldichlorosilane, 98:2 w/w) to prevent adhesion of the solid material to
the glass. The light source was maintained at a distance of 15 cm from the sample, routinely
for 20 s at room temperature.
1.9
Preparation of dityrosine standard and analysis
Dityrosine was prepared using a published method [6], with some modifications. L-tyrosine
(472 mg) was dissolved in 400 ml of boiling water. To this solution was added 100 ml of 0.5
M sodium borate buffer (pH 9.0). Following cooling to 37 ˚C, 10 mg of Arthromyces
ramosus peroxidase was added followed by dropwise addition with stirring of 35% hydrogen
peroxide to a final concentration of 5 mM. The solution turned light brown almost
instantaneously and was maintained at 37 °C for a further 15 min. The reaction was stopped
by addition of 175 l of -mercaptoethanol, and the sample was freeze-dried. The fluffy
white product was redissolved in 100 ml of water; the pH was adjusted to 8.8 and the solution
loaded onto a DEAE-Sepharose column (3 X 20 cm) that had been equilibrated using 20 mM
NaHCO3 (pH 8.8). The column was eluted using 20 mM NaHCO3 (pH 8.8) and the flowthrough fractions were collected and checked for fluorescence (in 100 mM NaOH) using a
plate fluorimeter (BMG Polarstar) with excitation (300 nm) and emission filters (400 nm)
fitted. The fluorescent fractions (450 ml) were adjusted to pH 3.0 with trifluoroacetic acid
(TFA) and loaded onto a preparative-scale HPLC column (Phenomenex C-18, 100 Å pore
7
size, 15 m particle size; 2.0 X 25 cm) at a flow rate of 20 ml/min. The column was washed
with 0.1% TFA in water for 10 min followed by a gradient of 0– 8% acetonitrile in 0.1%
TFA over 30 min. The eluate was monitored at 280 nm and samples were tested for
fluorescence as described above. Two major peaks were observed from the A280 recording of
the eluate. The first peak eluted at 15 min and the second at 30 min. Only the second peak
was fluorescent and upon freeze drying, yielded 376 mg of dry white solid material
(representing a 79% theoretical yield). This material was dissolved in 0.1 M formic acid and
was analysed by analytical HPLC (part 1.10), which showed that it was fluorescent and
eluted at 3.6 min compared to a tyrosine standard which eluted at 2.8 minutes. Electrospray
MS of the purified dityrosine sample indicated a major species of mol. wt. 361.2,
corresponding to dityrosine. This sample was used as a standard for HPLC analysis of
dityrosine in HCl-digested samples of both uncrosslinked rec1-resilin and crosslinked resilin
material.
1.10
Analysis of dityrosine in rec1-Resilin samples
For determination of the ratio of dityrosine to tyrosine in rec1-resilin samples, standard
curves were constructed using authentic tyrosine and a purified dityrosine standard. A sample
of photocrosslinked rec1-resilin hydrogel, formed from a 200 mg/ml solution of purified
rec1-resilin, and a sample of pure uncrosslinked recombinant rec1-resilin were digested in 6
N HCl and analysed for dityrosine content by HPLC. A sample (2 to 5 mg) of resilin was
weighed into a 4-ml screw-cap HPLC vial. To the sample was added 1 ml of 6 N HCl
containing 0.1% phenol, and the vial was sealed with a silicone-backed teflon coated septum
and two hard-plastic septa. The vials were transferred to a pre-heated block at 145 °C and this
temperature was maintained for 4 h. The sample was then diluted to 10 ml with HPLC mobile
phase buffer (8% acetonitrile, 0.1% TFA). A 1.5-ml aliquot was transferred to an Eppendorf
8
tube that was centrifuged at 14,000 rpm for 15 min. A 1-ml aliquot was transferred to an
HPLC vial followed by 20 l of a 10 mg/ml solution of raffinose (the internal standard).
After evaporation to dryness, the solution was reconstituted with 100 l of water and
transferred to an HPLC vial containing a 300-l insert for HPLC analysis.
Analysis of dityrosine was carried out with a Waters HPLC system comprising a 510 or 600E
pump, a Waters 717 plus autosampler, a Waters 474 fluorescence detector (ex, 300 nm; em
400 nm) and 486 UV detector. The Waters Empower chromatography system was used with
a flow rate of 1.5 ml/min, with an Alltech Alltima C18 column (4.6 x 150 mm) fitted with a
guard column. The injected volume was 10 l. The solvent system was 8% acetonitrile
containing 0.1% TFA.
The results of this experiment showed that approximately 21% of the total tyrosine was
recovered as dityrosine from the crosslinked rec1-resilin sample, compared to 0.56%
dityrosine recovered from a sample of uncrosslinked rec1-resilin. There was also a small
fluorescent peak eluting at 4.34 min which may correspond to orthodityrosine or trityrosine.
2.
Time course of dityrosine crosslinking
The effect of irradiation time on dityrosine crosslinking and stiffness of the crosslinked rec1resilin were investigated. For this experiment, a solution of rec1-resilin protein was irradiated
in PBS buffer in the presence of 2 mM [Ru(bpy)3]Cl2, 10 mM ammonium persulfate for
varying lengths of time. The samples of rec1-resilin solution were irradiated and the samples
then analysed for both dityrosine content and for resilience testing and penetration via SPM,
as described. The results are shown in Fig 1, below.
9
25
A
Dityrosine (%)
20
15
10
5
0
0
50
100
150
200
250
Exposure Time (s)
Penetration (nm)
400
B
350
300
250
200
0
50
100
150
200
250
Exposure Time (s)
C
96
Resilience (%)
94
92
90
88
86
84
82
0
50
100
150
200
250
Exposure Time (s)
Figure 1. Time course studies of rec1-resilin showing changes occurring on exposure to a
600-W tungsten-halide source. (A). level of dityrosine showing that a plateau is reached at
about 20%. (B). Penetration of an SPM tip (k =0.12 N/m) into the samples. The penetration
is inversely proportional to the modulus and shows the material getting stiffer with time of
exposure. (C). Resilience increasing to a plateau initially but decreasing at long exposure
times, suggesting some change to the network, even though this is not reflected in the
dityrosine level or modulus.
10
3.
Scanning probe microscopy (SPM) study
A rod (1.0 mm x 50 mm) of rec1-resilin was cast (200 mg/ml of rec1-resilin in 50 mM
Tris/HCl pH 8.0, 50 mM NaCl, 2 mM [Ru(bpy)3]Cl2, 10 mM ammonium persulfate) and
photochemically crosslinked (600 W at 15cm for 20 s). In a separate experiment, strip
samples (5 cm x 7 mm x 1 mm) were prepared using the same conditions. The rod was
attached to a metal disc using a small amount of nail varnish, whilst the flat strip samples
adhered of their own accord. The synthetic rubbers, butadiene (BR, Kosyn BR01) and
chlorobutyl (CIIR, 1068), were supplied by Bridgestone NZ as unfilled resilience buttons,
prepared using standard sulphur/zinc oxide/organic accelerator formulations and cured at
145°C for 24 min.
A Digital Instruments Dimension 3000 SPM was operated in force-volume mode using a
probe with stiffness selected to match the stiffness of the sample. Standard silicon nitride
probes with a nominal spring constant of 0.12 or 0.58 N/m were used for rec1-resilin and
native resilin samples. These samples were characterised in a phosphate-buffered saline bath
at a strain rate of 1Hz. For synthetic rubbers, silicon probes with a nominal spring constant of
50 N/m were used and the material was characterised in air. Typically, at least 3 forcevolume plots (16 x 16 arrays of force-displacement curves taken over a 10 x 10 m area)
were recorded with each of the samples. Resilience is a measure of a material’s ability to
recover after deforming under an applied stress. It is defined as the energy recovered after
removal of the stress divided by the total energy of deformation. The energy of
deformation/recovery is given by the area under the force-penetration/retraction curves.
Resilience was determined by converting force-displacement curves into forcepenetration/retraction curves and calculating the ratio of the areas under the penetration and
retraction curves [7].
11
4.
Tensile testing
Tensile tests were carried out on rec1-resilin in buffer on an Instron Tensile Tester
(model 4500) at a rate of 5 mm/min and a temperature of 21°C. The swollen strip samples
(7mm x 1mm) had a gauge length of 5 mm and were cycled initially up to a strain of about
200%. The maximum strain was increased successively in steps of 25-50% until failure
occurred (Fig. 2). Resilience was calculated from the ratio of the area under the retraction
curve to the area under the extension curve. The stress at 100% strain on the extension curve
was taken as the secant modulus at 100%.
The stress strain response of a rubber can be analysed according to the statistical theory of
rubber-like elasticity in order to determine the crosslink density of the material [8,9]. For a
swollen rubber the relationship is:
  NRT
1
3
2M c  
1 

* 1 
 *   2 
M  
 

(1)
where  is the stress (force per swollen, unstressed cross-sectional area), N the crosslink
density (equal to /Mc), R the gas constant, T the absolute temperature, the volume fraction
of rubber in the swollen sample (0.2),  the unswollen density (1.33 g/cc), Mc the molecular
mass between crosslinks, M the primary molecular mass (28492 g/mol) and  the extension
ratio. For an ideal elastomer, a plot of  verses ( - 1/2) should yield a straight line with a
slope that can be used to determine the mass between crosslinks (Mc). Figure 3 shows the
stress strain data for the extension (up to 100% strain) part of the first cycle, plotted
according to equation 1. Whilst the first section is reasonably linear with a slope of 733 Pa,
the data deviates from a linear relationship after (-1/2) reaches about 0.6 (25% strain). This
12
is slightly sooner than found by Weis- Fogh for natural resilin who showed the stress strain
data deviating from the theory at about 50% strain [10]. On average the slope of the stress
verses (-1/2) plots was 814 Pa which results in an Mc of 14161g/mol. Note that the value
of Mc is not very sensitive to the slope of the curve in Figure 3. Even if we double the value
of the slope the value of Mc only decreases to 14077 g/mol. Note too that the term (1-2Mc/M)
accounts for chain ends which do not contribute to the load bearing capacity of the material.
For most rubbers, both natural and synthetic, the primary molecular mass (M) is of the order
of 105 or 106 g/mol and (1-2Mc/M) is close to 1. In the case of the rec-1 resilin the relatively
low primary molecular mass results in a lot of chain ends and may be a major reason for the
lower than expected modulus.
13
8
Stress (kPa)
6
4
2
0
0
100
200
300
Strain (%)
Figure 2. Typical stress strain plots for a strip of rec1-resilin tested in buffer. Sample cycled
to 225%, showing resilience of 97% (pink curve) and later tested to failure showing
extension at break of 313% (blue curve). Also shown for comparison is the theoretical curve
(equation 1) using Mc =14161g/mol (green curve).
2.5
Stress (kPa)
2
1.5
1
y = 0.7332x - 0.0187
R 2 = 0.9885
0.5
0
0
0.5
1
1.5
2
( -1/  )
2
Figure 3. Stress strain data from the extension part (up to 100% strain) of the first cycle of a
test, plotted according to equation 1. The straight line is the least squares fit of the data up to
20% strain (-1/2 = 0.5).
14
5.
Expression profiles of the Drosophila resilin gene CG15920
Primers for quantitative real-time RT-PCR were:
Primer name
Res 2 Fwd (CG15920)
Res 2 Rev (CG15920)
Dros.RPLPO Fwd
(CG7490)
Dros.RPLPO Rev
(CG7490)
Sequence
CAGAACCAAAAACCATCAGATTC
GGCGGGCTCATCGTTATC
CTTCATCAAGGTTGTGGAACTGT
Size
23
18
23
Tm [˚C]
52
52
53
19
49
TTGGTGAACACGAATCCCA
Larvae, pupae and adult Drosophila melanogaster (Oregon-R strain; 50–100 mg) were
obtained from cultures maintained at the Department of Entomology, University of
Queensland, and were used for extraction of total RNA. Larvae were collected on days 4
through 8, including wandering larvae (LW), and stored in liquid nitrogen. Pupae were
collected immediately upon pupation (P1), and after several days when they had contracted in
length but were still light in colour (P2); dark coloured pupae were collected just before
emergence (P3). Adult flies (A) were collected immediately post-eclosion.
RNA was extracted by homogenisation in TRIZOL™ extraction reagent (Invitrogen), treated
with DNase (Ambion), and then passed through RNeasy RNA isolation columns (Qiagen) as
a second round RNA clean-up procedure with an additional on-column DNase treatment
(Qiagen).
Purified RNA (determined spectrophotometrically) was reverse transcribed essentially
according to the Superscript (Invitrogen) protocol. A minus RT control was included for each
tissue type to demonstrate in quantitative PCR that DNA contamination was negligible or
within acceptable limits (at least 12–15 cycles difference in detection). Reaction mixtures
containing NNdT20 oligonucleotide (2 g) or random hexamers (500 ng), total RNA (5 g)
and RNAsin (40 units, Promega) were heated at 70 ˚C for 10 min, then cooled in ice. Next,
15
reaction mixtures (50 µl) were made up to the composition of RT buffer containing 10 mM
dithiothreitol, 0.5 mM dNTPs, and heated to 42°C (oligo dT) or 37 °C (hexamers) for 2 min
before reverse transcriptase (200 units, Superscript) was added. After 1 h at 42°C (oligo dT)
or 37 °C (hexamers), the reaction was terminated by heat treatment at 70 °C (10 min).
The cDNA product was diluted 10-fold and used in quantitative real-time PCR analysis as
follows: To water (1.5 l) were added SYBR-Green master mix (Applied Biosystems, 2.5
µl), Fwd and Rev primers (450 nM, 0.25 l each), and diluted cDNA template (0.5 l). Four
technical replicates were conducted for each biological sample, and analyses were performed
in a 7900HT Sequence Detection System (Applied Biosystems) under the following
conditions: 95 °C for 10 min (Amplitaq Gold activation, 1 cycle), followed by 40 cycles of
95 °C for 15 s, and 60 °C for 1 min. Upon completion of the amplicon detection assay, a
dissociation analysis was carried out to ensure only a single amplicon species had been
generated.
Data were analysed using a specialised EXCEL program (Q-Gene,
www.Biotechniques.com). Data were normalised to a reference gene (ribosomal protein
RPLPO). The results indicated that resilin is expressed only in the pupal stages of
development; thus it seems not to be renewed during the life of the insect, and therefore must
have considerable fatigue resistance.
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