Cultures o f pFABhCGB and pFAB3 in E.coli strain 801-H were grown and induced under
difierent conditions (Figure 3.11). The western blot from these cells showed a very strongly induced band of 43kD in all the pFABhCGB and pFABhCGBAss post-induction lanes, though this varied in amount under the different conditions. The pFABhCGB cultures produced more glUp/hCGB when higher amounts o f IPTG were used for induction until a maximum was produced with 2 mM. Growing the cultures for longer than one hour post-induction only increased glUp/hCGB production when 2 mM IPTG was used. The pFABhCGB culutures grown at 30°C and induced with 2 mM IPTG produced the most fusion protein.
The pFABhCGBAss cultures did not show such a variation with the different culture conditions. There was httle difference between the cultures induced with 2 mM IPTG and with 1 mM, in both production increased shghtly after two hours. The culture grown at 30°C produced the same amount o f glUp/hCGB as the 2 mM culture, though it was produced maximally after only one hour.
Therefore it appears that 2 mM IPTG is sufi&cient for maximal induction and it is better to grow the cultures at 30°C. This probably further reduces proteolysis o f the foreign fusion protein.
Phage preparation ofpFABhCGfi and pFABhCGfiAss in E.coli strain 801-H and analysis hy Radioimmunoassay
Recombinant phage were prepared from pFABhCGfi and pFABhCGAss cultures to
investigate whether the hCGfi was e?q>ressed on the phage surface. The phage supernatant were analysed by RIA (Figure 3.12). None o f the phage preparations conq)eted with I^^^CG for binding to a rabbit polyclonal anti-hCG (P3). Therefore hCGfi was not detectable on the surface o f the phage.
Panning o f phage
In view o f the absence of detectable levels o f surface hCGfi in the total phage preparation, atterr^ts were made at enriching the phage population for any phage which might be expressing hCGfi.
Phage preparations were panned on tubes coated with P3 or with a rabbit polyclonal raised against thyroglobulin (Tg) as a negative control. Separate control tubes were coated with P3 or Tg antisera and incubated with I^^^CG. The I^^^CG control tubes had 148 counts for the anti-Tg coated tube and 12244 counts for the P3 coated tubes. This shows that the P3 antibodies were specific for hCG and were coated adequately.
Eluted phage were titrated. 2 x 10^^ cfu/ml (colony forming units, i.e. phage with pFAB
DNA and thus able to rescue untransformed E.coli on anq)icilllin plates) of
pFABhCGfiAss had been added initially. The titration o f the phage eluate as pfu (plaque forming units i.e. total number o f phage) showed no difference between the control tubes and the hCG coated tubes (Table 3.1).
No phage had specifically bound to the hCG antiserum, therefore it appears that there is no hCG on the surface o f the phage.
pFABhCGfiAss on anti-hCG coated tube on anti-Tg coated tube
1.5 X lOVml cfu 2.2 X 10^/ml cfu
4 X loVmlpfii 5 X lOVml pfu
Table 3.1 Titres of eluted phage expressed as both cfu and pfu.
The same number of phage wereeluted from the anti-hCG coated tube as from the anti-Tg coated tube. There were > 1 x lO^Vml pfu in the unbound supernatant from 2 x 1 0^^ cfu added.
Commercial helper phage used in preparation o f recombinant phage
The pHEN system has been used successfully in many laboratories. To estabhsh if there was a fault in our protocol we prepared bacteriophage following guidelines from Dr Greg Winter, who developed pHENl. The main difference in the method is that it uses helper phage M 13K07 which are purchased (Stratagene, California, USA) rather than grown in the laboratory.
These bacteriophage were concentrated 10 x by PEG precipitation and tested in an ELISA. They were captured with a polyclonal anti-hCG (P3) and detected with a
polyclonal anti-M13. If concentrated phage were used there was a high background signal (data not shown). This background was minimised if the concentrated phage were diluted
10 X (Figure 3.13).
When the mAh OT3 A was used for detection o f bound phage, no signal was observed. This could be explained in two ways: a) The C-terminus is next to glUp, perhaps the
epitope is not accessible to the antibody due to obstruction by the gHIp (Lowman et a/.,
1991) b) The anti-M13 is raised against phage coat proteins IH and V m and so there are a large number o f potential binding sites on each phage giving an amplification not possible with mAh 0T3 A. 0T3A can only bind to a maximum o f three to five fusion proteins per phage. This inphes that there was only a low percentage o f phage expressing hCGfi in the population.
pFAB3
ir
pFABhCG - 4 3 k DI
I
1 2 3 4 5 6 7 8 9 10 11Figure 3.10 Induction of pIII/hCG fusion protein in 801-1.
pFAB 3 and pFABhCGB in E.coli 801-1 were grown until O.D. 0.5, then samples harvested after 60 min (lanes 2 and 7), 95 min (lanes 3 and 8), 135 min (lanes 4 and 9), 240 min (lanes 5 and 10) and 305 min
(lanes 6 and 11). 10^ cells were run per lane on a 12% SDS PAGE gel, 2pg hCG protein was run in lane
1. The gel was transferred to nitrocellulose and blocked with 5% milk powder. SighCG (pre-adsorbed on an E.coli column) was incubated in the presence of E.coli lysate. After washing a peroxidase conjugated anti-rabbit polyclonal antiserum was used for detection and visualization was achieved with ECL and a 2 minute exposure. A band o f 43 kD was observed at all time points in the pFABhCGB, this band was absent in pFAB 3.
pFAB3 I pFABhCG pFABhCG s s
I
-— I
43kD • • • • -43kDH
to H 0 1 2 1 2 1 2 0 1 2 1 2 1 2 1 2 H 0 1 2 1 2 1 2 1 2IniM 2mM lOmM linM 2iiiM lOmM 30 ImM 2mM lOmM 30
Figure 3.11 Expression of hCGB from pFAB3 in 801-H.
Cultures of pFAB3, pFABhCGB and pFABhCGB ss in E.coli 801-H were grown to O.D. 0.4 and induced with different amounts of IPTG. 10^ cells were lysed and loaded per lane on 12% SDS PAGE gels from time 0 (uninduced), 1 (60 minutes) and 2(1 2 0 minutes after induction). Samples of pFABhCGB and pFABhCGB ss grown at 30°C were also run. The gels were transferred to nitrocellulose and blocked with 5% milk powder. The filters were incubated with SighCG (pre-adsorbed on E.coli
column) in the presence of E.coli lysate and detection was with an HRP conjugated anti-rabbit polyclonal antiserum. The results were visualized with ECL (3 minute exposure). H = hCG protein
<u CL 12000 10000 «000 WT pFABhCGB pFABhCGB ss 6000 4000 2000 250 30 66 125