All reagents were purchased from Sigma‐Aldrich Company Ltd (Dorset, UK), unless
otherwise stated.
6.2.1 Purification of 20S Proteasome from Human Liver
20S proteasome from human liver was purified, characterised, and used to degrade
insulin and a series of PEGylated insulins, from adapting previously described methodology
to purify 20S proteasome from animal liver (Bardag‐Gorce et al., 2004; Beyette et al., 2001;
Conconi et al., 1998; Conconi et al., 1996; Di Noto et al., 2005; Farout et al., 2006; Friguet et al., 2002; Rivett et al., 1994).
6.2.1.1 Liver Homogenisation and Centrifugation
Human liver (10 g starting material) was minced in 100 mL 50 mM tris‐HCl,
containing 250 mM sucrose and 150 mM NaCl, pH 7.5. Liver pieces were homogenised
using a TissueRuptor (Qiagen). The resulting homogenate was centrifuged at 10,000 g for
20 minutes at 4⁰C. The homogenate supernatant was removed and further centrifuged at
100,000 g for 1 hour at 4⁰C. The 100,000 g supernatant was removed and further
centrifuged at 100,000 g for 5 hours at 4⁰C. The resulting 100,000 g/5 hour pellet was
resuspended in 5 mL 20 mM triethanolamine buffer, containing 150 mM NaCl and 15%
(v/v) glycerol, pH 7.7.
6.2.1.2 Ammonium Sulphate Precipitation and Dialysis
Solid ammonium sulphate was added slowly to the 100,000 g/5 hour fraction to
give a 35% saturated solution, at 4⁰C, and left stirring for 1 hour, followed by a 15 minute
centrifugation at 10,000 g (4⁰C). The resulting supernatant was removed and further solid
stirred for 1 hour, re‐centrifuged and the resulting pellet resuspended in 2 mL 20 mM
triethanolamine buffer. The sample was then dialysed against 1 L triethanolamine buffer
(MWCO 12 – 14,000) overnight at 4⁰C, with one buffer change.
6.2.1.3 Anion Exchange Chromatography
The ammonium sulphate dialysed fraction was loaded onto a DEAE 5PW anion
exchange column (Waters) equilibrated in 10 mM tris‐HCl, pH 7.2, and proteasomes eluted
using a linear gradient of NaCl from 0.1 – 0.5 M over 30 minutes, during which fractions (1
mL) were collected. Active fractions containing specific proteasomal activity were pooled,
concentrated and loaded onto a Mono Q HR 5/5 column (GE Amersham) equilibrated in 10
mM Tris‐HCl, pH 7.2, and proteasomes again eluted using the same, linear NaCl gradient.
Active fractions were pooled and concentrated and protein content determined by the
Bradford assay.
6.2.2 Proteasomal Activity Assay
To a 96‐well plate, 50 µL of each anion exchange fraction was added to 50 mM
Hepes containing 50 µM boc‐LSTR‐AMC (prepared from a 10 mM stock in DMSO) in a 200
µL final volume, pH 7.8. Plates were incubated for 1 hour at 37⁰C, after which the released
fluorescence from each fraction was determined with Ex/Em wavelengths of 360 and 460
nm, respectively.
6.2.3 Proteasomal Degradation of Insulin and PEGylated Insulin in vitro
Insulin and PEGylated insulin were incubated ± 20S proteasome, at the stated
amounts, at 37⁰C in 50 mM Hepes containing 5 mM DTT, 100 mM NaCl, 1 mM MgCl2, pH 7.8, in a 60 µL final volume.
6.2.3.1 Western Blot Analysis
Aliquots (10 µL) were removed at the stated times, diluted with ddH2O and stored at ‐80⁰C prior to SDS‐PAGE/western blot analysis to assess degradation, as described in
section 2.2.7.
6.2.3.2 LC‐ESI‐MS/MS Analysis
The peptide repertoires generated following proteasomal degradation were
analysed by MS as described in section 5.2.3.2.; incubations were for 12 hours, however.
6.3 RESULTS
A 20S proteasome fraction was obtained from human liver using adapted
methodology. This fraction was shown to be highly purified and capable of degrading
protein. These proteasomes were subsequently used to assess the effect of PEGylation on
the proteasomal degradation of insulin, a model protein.
6.3.1 Purification and Characterisation of Human Liver 20S Proteasome
Following homogenisation and differential centrifugation of human liver, the pellet
was further purified using ammonium sulphate precipitation and dialysis. After dialysis, the
resulting fraction was further purified using two ion exchange columns. Fractions were
assessed for specific proteasomal activity using two fluorogenic probes, boc‐LSTR‐AMC and
suc‐LLVY‐AMC, which assess the chymotrypsin‐like and trypsin‐like activity of the
proteasomes, respectively. In their native state, the fluorophore AMC is quenched due to
the attachment of the bulky blocking groups; succinyl (suc) and N‐t‐butyloxycarbonyl (boc).
Cleavage of the peptide, linking AMC to the quenching group, by the proteasome results in
the release of the fluorescent AMC, which can be measured fluorometrically. Thus, the
content. Following the second ion exchange step, active fractions were pooled,
concentrated, and the protein content determined by the Bradford assay. To assess the
purity of the final 20S proteasome fraction, 5 µg of protein from the fraction was resolved
on an SDS‐PAGE gel, alongside 5 µg of protein taken from earlier fractions, and stained with
coomassie, as shown in figure 6.1. In figure 6.1, the final 20S fraction was resolved in lane
G. In this lane the only protein bands visible are between ~22 and 35 kDa, which are the
Figure 6.1: Purification of 20S proteasome from human liver
Protein (5 µg) from each fraction was loaded onto a 12% SDS‐PAGE gel and stained with
coomassie following electrophoresis. Lanes – A: Homogenate supernatant, B: 100,000 g/1
hour supernatant, C: 100,000 g/5 hour pellet, D: Ammonium sulphate dialysed fraction, E:
DEAE (100,000 g/5 hour pellet) active fractions, F: DEAE (Ammonium sulphate dialysed
fraction) active fractions, G: Mono Q (DEAE combined) active fractions. Bands highlighted
in Lane G are the characteristic proteasomal subunits.