Download Role of Proteus mirabilis metalloprotease in degradation of different

) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
University of Baghdad
College Name
College of science
Department
Department of biology
Full Name as
written
inPassport
e-mail
Dr Bahaa Abdullah Laftaah Al-Rubai
[email protected]
Career
Assistant Lecturer
Master
Thesis Title
Year
Lecturer may
Bahaa
Abdullah
Laftaah AlRubai
AssistantProfessor
Professor
phD Bahaa Abdullah Laftaah Al-Rubai
Role of Proteus mirabilis metalloprotease in
degradation of different animal proteins and
cloning of responsible gene in
E
.coli BL21
2009
) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
I
Summary
Abstract
1.
The clinical strain of Proteus mirabilis 49118-3 causes Urinary
Tract Infection was obtained from China Gene Bank; this strain was
ability to hydrolyze casein of skimed milk agar (clear zone around the
colonies). Different liquid media containing various pertinacious
compounds included peptone, tryptone, yeast extract and tryptic soy
were used, Luria-Bertani (L.B) broth which contain (tryptone and yeast
extract) gave the highest protease specific activity 145 unit/ mg protein
when it was inoculated with P. mirabilis and incubated for 48hr at 37°C
in shaking incubator with 180 rpm.
The metalloprotease P. mirabilis was purified by precipitation with
60% saturation of ammonium sulfate, dialysis and gel filtration with
Sephadex G-100 column, three peaks of protein appeared in last step,
the protease activity was observed in third peak.
The molecular weight of P. mirabilis metalloprotease was
Daltons. 44755determined by SDS-PAGE it was approximately
P. mirabilis metalloprotease was treated with different therapeutic
agents included: anti-swarming agent and antibiotics to determine their
effect on the activity of protease. The anti-swarming agent pNitrophenylglycerol (PNPG) inhibited the protease activity at
concentration 10-500 µg. whereas the antibiotics varied in their effect,
some of them decreased protease activity such as kanamycin ,
streptomycin, penicillin-G, erythromycin and tetracycline, the remaining
activity of the enzyme treated with 75µg/ml of these antibiotics was 73
, 62 , 55, 35, and 22 % respectively, while the ampicillin revealed
) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
different effect depending on its concentrations since, the activity of
enzyme increased to 107 , 125 % at 25 and 50 µg/ml respectively, and
decreased to 72% at 75µg/ml of ampicillin.
The effect of P. mirabilis metalloprotease on the human ß-defencen
1 (hBD-1) was studied; the bactericidal activity of this proteins was
inhibited after treatment with P. mirabilis metalloprotease against
Escherichia coli 0138 for 2 hours.
In this study the metalloprotease of P. mirabilis revealed cytotoxic
activity on monolayer of Vero cells which completely damaged after 7296 h and causing death of the cell culture, the enzyme was able to
degrade different animal cell matrix compounds including collagen IV,
elastin, laminin and fibronectin, also the metalloproteases able to
hydrolyze other anmals proteins such as hemoglobin, gelatine, and
bovin serum albumin (BSA) as well as casein.
Zap A gene encoded P. mirabilis metalloprotease was amplified by
polymerase chain reaction technique (PCR) and cloned in pGEX-KG
plasmid to construct new recombinant pGEX-KG (ZapA) expression
vector and transformed into E.coli BL21 DE3 to overexpression the
recombinant metalloprotease , E.coli failed in expression of the cloned
gene.