4.1 NIVEL DE CONOCIMIENTO SOBRE LOS ESTUDIOS CRÍTICOS
4.1.1 INTERPRETACIÓN DE LA INFORMACIÓN-ENTREVISTA
It is estimated that 2.5mg of purified protein could be obtained from 1 litre (I) of bacterial culture using the GST-system (GST Gene Fusion System
instructions booklet, p. 11). For the large-scale production of GST-utrophin fusion proteins II or 21 culture volumes were routinely used. This procedure is described in detail in section 2 . 2 and as an “aide mémoire” is shown
diagrammatically in Fig 3.10.
The purified proteins and fractions collected at different stages of the purification procedure were analysed by SDS PAGE and Coomassie Blue staining. The utrn-3 preparation is shown as an example in Fig. 3.11. Lane 1 represents the total protein content of the sonicated bacterial culture; the SOkDa band corresponds to GSTutrn-3 fusion protein. The majority of GSTutrn-3 remains in the supernatant after centrifugation (lane 2) but some is retained in
the insoluble pellet (lane 3). Lane 4 is the supernatant after filtering through a 0.45pm filter and immediately prior to adding to the GST affinity column. The SOkDa polypeptide constitutes a major component of this fraction,
approximately 5% of the total protein.
50ml of fraction 4 were applied to the GST affinity column and recycled twice. A small proportion of GSTutrn-3 apparently did not bind and was
collected in the first PBS wash (lane 6) but no further GSTutrn-3 was collected
in the second and third washes (lanes 7 and 8). 2.5pl and 5 pi of a 1 in 10
dilution of the utrn-3 eluate (total volume SOOpI) after thrombin digestion are shown in lanes 11 and 12. After thrombin cleavage the protein designation loose their GST component and become utrn-1 to 4. Thrombin digestion yielded
bacterial culture in BL21 IPTG
I
1:100 32°C 3hr 32°C V 4hr 8K rpm lOmln 2ml X 5 200ml x 5 200ml X 5bacterial cells cell extract
Ix P B S sonication 10 sec 10ml X 5 +Triton SOmin wash Ix P B S thrombin collect o/n rt m m m
□
+ □
□ 10K rpm 10min 0.45pm filter #. sepharose > glutathione □ GST □ utrophin protein* unrelated bacterial protein
Fig 3.10 Diagrammatic representation of a large scale preparation and purification of GST-fusion proteins, o/n =overnight; rt =room-temperature; PBS=phosphate buffered saline; Triton=Triton X-100.
66kDa 56kDa 43kDa- 36kDa. 26kDa- 'Hb
#
I
-60kDa -SOkDa .40kDa -30kDa M1 12 11 10 9 8 7 6 5 4 3 2 1 M2Fig 3.11 Large scale preparation of utrn-3. 2.5 pi and 5 p i of purified utrn-3 (lanes 12 and 11) and purified thrombin protein (lanes 10 and 9) and 10 p I of a series of fractions collected at different stages of the preparation (lanes 1 to 8)
were separated on a SDS PAGE and stained with Coomassie blue. M l lane and M2 lane contain 20ul of New England Biolabs and Gibco BRL mw markers respectively.
a utrn protein of the correct mw of c. 24kDa. The calculated concentration of affinity purified utrn-3 was estimated spectrophotometrlcallly as ôpg/pl. 2.5pl and 5pl of a 1 In 10 dilution of the purified thrombin protease protein were also loaded In the gel (lanes 9 and 10) to check whether these dilutions could be detected by Coomassie blue staining and whether they could contribute a band to those of the final eluate. As It turns out, these amounts were almost
undetectable and did not appear to co-purIfy with utrn-3.
The same procedure was used to generate GSTutrn-1 and GSTutrn-4 fusion proteins. The concentrations of the affinity purified thrombin cleaved proteins were lOmg/ml for utrn-1 and 2.5mg/ml for utrn-4. Fig 3.12 shows 5 pg of each purified protein run on a SDS PAGE and stained with Coomassie blue; utrn-3 with a mw of 24kDa and utrn-4 with a mw of c.36kDa. Utrn-1 comprises two bands of 36kDa and 32kDa (the estimated mw for utrn-1 Is 36kDa). Since the two bands appear only after thrombin digestion and were not apparent In the small-scale experiment (Fig.3.5), It was thought that there might be a weak recognition site for thrombin within the utrophin aa sequence. Examination of the aa sequence found a sub-optimal recognition motif (R K L L) 44 aa
downstream of the thrombin cleavage site In the vector (the optimal sequence Is P K L P2 where P2 Is a non-acldic aa). If the sub-optimal sequence was being
recognised by thrombin the mw difference between the two fragments would be 5kDa whereas the mw difference seen on Western blots Is around 4kDa.
In order to Investigate this further, the 36kDa and 32kDa bands from 5 pg of utrn-1 were separately excised from a SDS PAGE gel and eluted with PBS. The Isolated bands were electrophoresed alongside a sample of utrn-1 which
-40kDa
. SOkDa
Fig 3.12 Purified utrn proteins after GST affinity chromatography and thrombin cleavage of the GST-utrn fusion proteins. M= mw marker (Gibco.BRL)
had not been eluted from a gel, and immunoblotted with two antibodies raised against the NH2-terminus of utrophin; DRP2 (aa 1 to 261; Novocastra) and
Mannut-1 (aa 113 to 371; Nguyen et al., 1995). The aa sequences used to raise these two antibodies overlap with that of utrn-1 (aa 24 to 320); therefore, both antibodies should detect both bands.
The results of this experiment were unexpected (Fig 3.13 A). Mannut-1 detected only the 36kDa band while DRP2 detected the 36Da and 32kDa bands. Interestingly, in the lane loaded with isolated 32kDa band lane, two bands of 36kDa and 32kDa are seen instead of a single 32kDa band. In Fig 3.13 B a second experiment was carried out; SOOng of utrn-1 were loaded and in this case Mannut-1 detected exclusively the 36Da band and DRP2 detected both the 36kDa and 32kDa bands. These experiments suggest that the two bands may represent two “conformations” of the same utrophin polypeptide.
The most likely explanation is that the 32kDa represents an intermediate “folded” form of GST-1 which cannot be detected by Mannut-1 because the aa sequence involved in the fold coincides with the binding site of Mannut-1, which must lie somewhere between aa 113 and 340. However, DRP2 interacts with a sequence, somewhere between aa 24 and 261 in utrn-1, that is available in both the “folded” and “unfolded” states. There is also the conversion of the fast mobility form “32kDa” to the “36kDa” form during electrophoresis (Fig 3.13 A lane 32kDa). It is difficult to explain how the 32kDa fast mobility polypeptide retains some structure in the presence of 0.5% SDS denaturing gel and
suggests a very stable folding intermediate. Unfolding of most proteins is a two stage process with at least one folding intermediate involved (Creighton, 1997)
36kDa— ^ 3 2 k D a _ ^ 24kPa . ^
C 36KDa 32kDa S. 36KDa 32kDa c 36KDa 32kDa g --- g g --- W utrn-1 I I (1^ utrn-1 I I J utrn-1
Mannut-1 DRP2 Urd40 5. 36KDa 32kPa & utrn-1 I 2nd antibody
II
} }
CO - k Mannut-1 PRP2 -36kPa Mancho-3Fig 3.13 Western blots of purified proteins using utrophin specific monoclonal antibodies.A, the lower mw (32kDa) and higher mw (36kDa) bands that appeared after digestion of GSTutrn-1 with thrombin were run separately and probed with Mannut-1 and DRP2. utrn-3 was also run in this gel and was detected with Urd40. Incubation with second antibody failed to detect any band and confirmed the specificity of the antibody-antigen interactions. B, 500 ng of purified utrn-1 and utrn-3 probed with Mannut-1 and DRP2. C, purified utrn-4 and utrn-1 probed with Mancho-3.
and indeed monoclonal antibodies are used to study the conformational fluctuations of proteins (Creighton, 1997). There are several factors that may affect the shape of proteins on a SDS gel (and their electrophoretic mobility) and these include; presence of disulfide bonds, high proline content and very high or very low isoelectric points. An internal disulfide cannot form as there is only a single cysteine in utrn- 1 ;the proline content is not remarkable and the
isolectric point is 6 . 0 (neutral); so it is not obvious what factors lead to utrn- 1
folding.
To examine the specificities of utrn-3 and utrn-4, they were
immunoblotted with the Urd40 and Mancho-3 antibodies which should detect utrn-3 and utrn-4 respectively. Exactly as might be expected Mancho-3 detected utrn-4 but not utrn-1 (Fig 3.13 0 ) and Urd40 detected utrn-3 but not utrn-1 (Fig 3.13 A).