Medición de las variables de calidad

Immunofluorescence studies using the anti-C4 monoclonal antibody (methods, section D. 1) were carried out on SV40 transformed 3T3 fibroblasts, cells known to express only the C4^ polypeptide (results section C; fig 2 g). Studies showed uniformly stained linear actin filament stress fibre bundles (fig 1 b and d). Both cell processes and diffuse actin networks, which are present in these cells, are also stained with anti-C4 (fig

t s 3 ^

?■

FIGURE 1. Immunofluorescence localisation in SV40 transformed 3T3 fibroblasts of

C4K Cells are shown under phase constrast (a and c). Immunofluorescence of the same

cells shows the superposition of anti-C4 monoclonal antibody (b and d). Uniformly stained linear actin filament stress fibre bundles can be seen (b) and in many cells, nuclear staining is observed (d). Bar= 10pm.

r . TTSSIIK SKt-FCTION FOR THF. PirRfFirATTON OF

C4

SDS-PAGE and immunoblot analysis of human T cell lymphoma (HTCL) and SV40 transformed 3T3 fibroblasts using an affinity purified rabbit anti-C4 polyclonal antibody (Shapland et al, 1993; methods, section D. 2) revealed that 04^ was present at high levels at a molecular weight of approximately 21KDa (fig 2 c, e and g). Immunoblotting of rat embryo fibroblasts (REF) indicated the molecular weights of C4^ and transgelin which are present in approximately equal amounts and are separated by 0.5.1 Kd in these cells (fig 2 c).

From these data HTCL were chosen for the purification of C4k These cells expressed higher levels of C4^ than both activated T cells and thymus. They were easy to grow in culture, growing rapidly and harvested without trypsinisation (unlike SV40 transformed 3T3 fibroblasts).

D. PURIFICATION OF C4i 1. Detergent Solubility of C4l

Figure 3 shows C4^ solubility in the zwitterionic detergent CHAPS. HTCL cells lysed in wash buffer + 1% CHAPS (fig 3 a), and the subsequent pellet (fig 3 c) and

supernatant (fig 3 e), derived after centrifugation were analysed by SDS-PAGE and immunoblotting with monoclonal anti-C4 antibody. Millipored (0.45jiM) HTCL 1% CHAPS lysate was also analysed to show loss of C4^ yield during the millipore process (fig 3 g). Figure 3 shows that the majority of C4^ was solubilised after HTCL lysis in 1% CHAPS.

2. Proteolysis of C4i

To minimise proteolytic degradation a cocktail of proteolytic inhibitors was used during all steps of purification. To show that these were effective in blocking C4^ degradation I dialysed an HTCL 1% CHAPS lysate for 50 hours at 4°c in the presence or absence of the proteolytic inhibitors listed in table 1 and analysed C4^ degradation over time by immunoblotting with the anti-C4 monoclonal antibody (fig 4).

9 7 4 6 3 43 25-7 18-4 14‘3

a

b

o

d

e

f

9

FIGURE 2. Im m unoblot analysis of proteins transferred to nitrocellulose m embranes labelled with a rabbit anti-C4 polyclonal antibody. M olecular w eight m arkers are shown

(a) with weights in kDa on the left. C4^ and transgelin are present (in approxim ately

equal amounts) when total REF lysate (b) is transferred to nitrocellulose (c). Total HTCL

lysate (d) expresses only the lower m olecular weight 04^ (e). Identical results are seen

-21

a

C

e

g

FIGURE 3. Detergent solubility of C4^: Total HTCL cells (a) were lysed in wash buffer + 1% CHAPS and the C4^ population shown when the lysate w as transferred to nitrocellulose (b) and probed with the anti-C4 m onoclonal antibody. The subsequent pellet derived from centrifugation (c) is shown to contain no detectable population o f C4^ when transferred to nitrocellulose (d). The resultant supernatant derived from the same centrifugation process (e) is seen to contain a C4^ population when transferred to nitrocellulose (f). There appears to be no significant loss of the C4^ population when a H TCL lysate centrifugation supernatant is 0.4 5 |im m illipored (g) and transferred to nitrocellulose (h). The arrow on the right indicates a m olecular w eight o f 21 kDa and the presence of C 4 l

Table 1. Protease Inhibitors. 'From Harris & Angal, 1989)

Inhibitor Target Effective W orking

C o n c e n tra tio n

Stock Solution Chymostatin

(Sigma)

Chym otrypsin 2ug/m l 5m g/m l in DMSO

(-20ÛC)

E DTA (Sigma) M etalloproteases 0.2mM 0.5M in H2O

pH 6.0 (RT) Leupeptin (Sigma) Serine and Thiol-proteases 2ug/m l 5m g/m l Sterile H2O (-2Q0c) Pepstatin A (Sigma)

Acid Proteases 2ug/m l 5m g/m l

in m ethanol (-20°c)

PMSF (Sigma) Serine Proteases 0.2mM 1 OOmM in

Isopropanol

Figure 4 illustrates that C4^ maintains its integrity for up to 50 hours when the proteolytic inhibitors are present. As the proteolytic inhibitor cocktail prevented C4^ degradation during cell lysis and dialysis, it was therefore utilised during all subsequent steps Qf purification at the same working concentration.

3. Chromatofocusing

Utilisation of a pH 7-9.6 chromatofocus column, employed directly after 1%

CHAPS lysis and dialysis in start buffer, extensively separated an HTCL lysate of typically 5x 10^ cells. This analysis revealed that C4^ eluted from the chromatofocus within a pH range of 8.0-8.2 in a concentrated peak obtained in 8 ml. SDS-PAGE analysis of 0.5 ml fractions within this peak are shown in figure 5. Four fractions within this peak (fig 5 h, i, k and 1), show C4^ as being the major protein in a fraction containing 11 visible bands. These fractions were pooled for further purification.

4. Hvdroxvlapatite Purification

A final purification and concentration step of the pooled chromatofocus fractions positive for C4^ was carried out on a small (3ml) hydroxylapatite column. Elution produced a preparation containing a band present in three fractions of 0.5 ml each (fig 6 f, h and j). The bands appeared to be higher than 90% homogenous with no apparent

degradation as shown by SDS-PAGE and immunoblotting (fig 6). These bands migrated at the same position in immunoblots as C4^ (seen previously in results, section C). By pooling the three fractions approximately 10|ig of > 90% homogenous C4^ was obtained.

■ Ml

0®in

g

4 — 21

FKilJRE 4. C4^ proteolysis: C4^ is seen to maintain its integrity when an H TCL lysate