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Figure 2.
FIGURE 2. Expression and activity of InpA. A, expression
and purification of pro-cd-InpA C154A. Lanes 1 and 2, E.
colihomogenate before and 3 h after protein expression
induction, respectively.Lane3, recombinant protein after
affinity chromatography purification. Molecular masses of the
distinct protein species (40 and 27 kDa) are shown on the left.
B, same for wt pro-cd-InpA. C, time-course analysis of
autocatalytic processing and activation of wt pro-cd-InpA (final
concentration, 10 µM) incubated with 1 mM HgCl[2]. The
reaction was initiated by adding EDTA (5 mM final concentration)
as an Hg^2+-chelator, i.e. by releasing metal-mediated
inhibition. Samples were withdrawn at the time intervals
specified (O/N, overnight incubation; lane C, pro-cd-InpA
alone). D, same as C but after addition of active InpA (10 nM
final concentration) to the reaction mixture. In this case, the
reaction proceeded much faster. E, a subset volume of the
withdrawn aliquots from C and D was used to quantify the
activity released from wt pro-cd-InpA in the absence (  ) and
presence (  ) of catalytic amounts
of wt cd-InpA. As a control, pro-cd-InpA spiked with InpA but
without EDTA was incubated in parallel (  ). F,
concentration-dependent autoactivation of pro-cd-InpA. The
reaction was initiated by releasing Hg^2+-mediated inhibition in
mixtures containing 0.04 µM ( ), 2 µM ( ), and 10
µM (  ) zymogen. At indicated
time points, 50 µl( ), 10 µl( ), and 2
µl( ) were withdrawn from
each reaction mixture and directly assayed for activity. G,
SDS-PAGE of the digestion of pro-cd-InpA C154A by wt cd-InpA.
The zymogen (final concentration of 10 µM) was incubated
with cd-InpA (0.1 µM) for time intervals as specified
(lane C, control pro-cd-InpA C154 incubated alone). N-terminal
amino acid sequences of pro-cd-InpA derived fragments are
indicated on the right. H, Western blot analysis of culture
supernatant of P. intermedia using InpA-specific rabbit
antiserum (lane 3). Wt cd-InpA and pro-cd-InpA (C154A) were
loaded on lanes 1 and 2, respectively, for comparison.
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