C. Chervaux et al., SECRETION OF ACTIVE BETA-LACTAMASE TO THE MEDIUM MEDIATED BY THE ESCHERICHIA-COLI HEMOLYSIN TRANSPORT PATHWAY, MGG. Molecular & general genetics, 249(2), 1995, pp. 237-245
An in frame gene fusion containing the coding region for mature beta-l
actamase and the 3'-end of hylA encoding the haemolysin secretion sign
al, was constructed under the control of a lac promoter. The resulting
53 kDa hybrid protein was specifically secreted to the external mediu
m in the presence of the haemolysin translocator proteins, HlyB and Hl
yD. The specific activity of the beta-lactamase portion of the secrete
d protein (measured by the hydrolysis of penicillin G), approximately
1 U/mu g protein, was close to that of authentic, purified TEM-beta-la
ctamase. This is an important example of a hybrid protein that is enzy
matically active, and secreted via the haemolysin pathway. Previous st
udies have indicated that haemolysin is secreted directly into the med
ium, bypassing the periplasm, to which beta-lactamase is normally targ
eted. This study indicated, therefore, that normal folding of an activ
e beta-lactamase, can occur, at least when fused to the HlyA C-terminu
s, without the necessity of entering the periplasm. Despite the secret
ion of approximately 5 mu g/ml. levels of the active beta-lactamase fu
sion into the medium, there was maximally only a 50% detectable increa
se in the LD(50) for resistance to ampicillin at the individual cell l
evel. This result suggests that, normally, resistance to ampicillin re
quires a high concentration of the enzyme close to killing targets, i.
e. in the periplasm, in order to achieve significant levels of protect
ion.