Page 11 - BM 2011 Vol.2 No.4

Bioscience Methods
BM 2011, Vol.2, No.4, 21-30 http://bm.sophiapublisher.com
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resultant heavy blue precipitate and the total protein
content of the clear supernatant determined by using
the BCA assay (Pierce Biotechnology, IL, USA).
Trypsin (Porcine, Sigma-Aldrich) was added at an
enzyme to substrate (Figure 1, supernatant 2) ratio of
1:20 to the concentrated supernatant and the solution
digested for 3 h at 37 . Digestion was halted by the℃
addition of benzamidine hydrochloride, EDTA (and
sodium azide) as indicated above and the solution
further concentrated to a final volume of 36 mL. This
strategy relies on the relative resistance of mucins to
tryptic digestion.
3.3 Hydrophobic interaction chromatography (HIC)
15 mL (packed column volume) of high substitution
Phenyl Sepharose (GE Healthcare USA) was used to
fractionate the soluble extract. The trypsin digested
concentrate was dialysed against 50 mmol/L sodium
phosphate (pH 7.0), 0.8 mol/L ammonium sulphate
and applied to the column in 6×6 mL aliquots. The
break-through fractions were collected, pooled and
bound proteins recovered from the resin with a 10 mL
pulse of 50 mmol/L sodium phosphate (pH 7.0).
SDS-PAGE was performed on all fractions collected
from the HIC column to confirm the presence of
trypsin-resistant proteins. Pooled break-through
fractions from the HIC purification were dialysed
against PBS and used for compositional analyses and
bacterial adhesion assays. The protein in the break-
through fractions stained strongly with Alcian blue
after SDS-PAGE and hence it was called bell mucin.
3.4 Protein extraction from external mucus-like
fluid (exudate mucin)
Nine hundred ml of external mucus-like fluid, released
from the jellyfish during collection, was thawed and
concentrated to 46 mL by using an ultra filtration YM
10 (Millipore Corp). The concentrate was dialysed
against 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L
sodium chloride and any precipitant removed by
centrifugation (70 000×g, 40 min, 4 ). The total℃
protein content of the supernatant was determined
using the BCA assay (Pierce Biotechnology), and the
sample was then digested with trypsin as described
above. Digestion was halted by addition of
benzamidine hydrochloride, EDTA (and sodium azide)
at final concentrations as indicated above and then the
sample was concentrated to 23 mL, dialysed against
phosphate buffered saline (PBS) and frozen at - 20 .℃
3.5 Amino acid and monosaccharide compositional
analyses
For amino acid and monosaccharide compositional
analyses, the HIC break-through fractions rich in
mucin-like protein were pooled, dialysed against
50 mmol/L TrisHCl (pH 8.0), 150 mmol/L sodium
chloride and further purified by gel permeation
chromatography (GPC) on a TSK-Gel G3000SW
7.5 mm×30 cm column (Tosoh Corporation Japan).
Fractions eluting in the void volume were collected,
pooled, concentrated and dialysed against distilled
water and amino acid analysis performed by the
Australian Proteome Analysis Facility Ltd.,
(Macquarie University, Sydney, Australia) using stan-
dard protocols (Sando et al., 2009). Cys and Trp
contents were not determined. Monosaccharide com-
positional analysis was determined by the Glyco-
technology Core Resource Facility, (University of
California, San Diego, USA) using chromatographic
techniques (Sando et al., 2009).
3.6 Bacterial culture
A corneal isolate of P. aeruginosa (strain Paer6294-
GFP) was used in the study. This strain was originally
isolated from an infectious corneal ulcer (Aristoteli
and Willcox, 2001) and then engineered to harbour a
plasmid expressing green fluorescent protein (GFP)
(Christensen et al., 2007). This allowed the detection
of bacteria by fluorescence. The strain was taken from
- 86 storage and subcultured once on chocolate℃
blood agar plates (Micro Diagnostics, Brisbane, QLD,
Australia) containing 200 µg/mL carbenicillin at 35℃
for 18 h. The day before use, bacterial colonies were
resuspended in 0.01 mol/L PBS (pH 7.4). They were
then centrifuged at 3 000×g for 10 min, washed once
with PBS, pelleted and resuspended in Minimum
Essential Medium Eagle (MEM, Life Technologies,
Grand island, NY, USA) supplemented with 0.6%
(w/v) bovine serum albumin (BSA, Sigma-Aldrich)
and 0.035% (w/v) NaHCO3.
3.7 Cell culture
Human corneal epithelial (HCE) cells were cultured
in modified SHEM (supplemented hormone epithelial