【For INTERNATIONAL COLLABORATIVE RESEARCH 2018】

DATE: DayMonthYear 2019

SUMMARY of2018 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University

NameInvestigation of Transcriptional Regulators as Potential Drug
Targets
Dr. Inoka C. Perera

Affiliation

University of Colombo

Present Title

Senior Lecturer

Research Title
ApplicantResearch Collaborator (Host PI)

Prof. Genji Kurisu

Summary
Pseudomonas aeruginosa is metabolically versatile gram negative, aerobic, motile, monoflagellated,ubiquitous, opportunistic pathogen predominant in the stratum corneum. They express virulence factorswhich enable bacteria to thrive in various environments such as soil, water, marine habitats and animal tissues
as well as in harsh environments such as hot springs, crude oils as they have very high adaptability.
Maintenance of inner and outer membrane is essential for bacteria for active life in harsh environments as it
is directly contacting with the environmental substances. Membrane fluidity is tightly regulated by regulating
the level of saturated and unsaturated fatty acids in their membrane bilayers. Because imbalance in membrane
fluidity is detrimental to the cells. Pseudomonas aeruginosa encodes two alkane monoxygenases annotatedas AlkB1 and AlkB2 with overlapping substrate range. AlkB1 and AlkB2 facilitate to uptake n-alkanes fromC 10 to C 24 . n-Alkanes convert into fatty acids via sequential oxidation of terminal methyl group via bacterial
metabolism. FadR type regulators have identified in regulation of expression of critical enzymes involved infatty acid biosynthesis and degradation either as a repressor or activator. PA1526 is a GntR family regulator
confine to FadR sub-class in P. aeruginosa where divergently transcribed from Alkane-1-monoxygenase 2(AlkB2) gene. Transcriptional regulator PA1526 in Pseudomonas aeruginosa is proposed as a potent drug
target to battle infections of P. aeruginosa as it regulates an important pathway responsible for the ubiquitous
existence of the organism. Three-dimensional (3D) structure of the target protein needs to be determined forstructure based drug discovery. Protein structural studies were performed to determine the model of PA 1526.
Recombinant PA1526 was purified to near homogeneity to screen crystal formation at different conditions.
However, Crystallization of PA1526 was not successful. Circular dichorism (CD) spectroscopy revealed PA
1526 has high number of α helixes and melting temperature of PA1526 is 40 ℃. Transcriptional regulation
is a fundamental biological process crucial for Mycobacterium tuberculosis for its survival in macrophages
upon various stress conditions including nutrient deprivation, hypoxia, acidic pH, reactive oxygen species,
reactive nitrogen species and antibiotics. Mycobacterium tuberculosis Rv0792 regulator protein, considered
to be responds to oxidative stress or in an important cell cycle pathway or antibiotic synthesis pathway.
As the last year work summary, the constructed pET 28a with Rv0792 gene was transformed into E. coli
BL21(DE3) for overexpression with IPTG. The expressed Rv0792 protein was purified using Ni-NTA

【For INTERNATIONAL COLLABORATIVE RESEARCH 2018】

chromatography and further purified using size exclusion chromatography following TEV protease cleavageof His tag. A final concentration of 5 mg/ml of protein was obtained with 90% homogeneity. Crystal screening
was carried out for this concentration using screening kits, Crystal screen I & II, Wizard I, II, III & IV andPEG/Ion I & II. The crystals observed under different conditions were confirmed as salt crystals. CD spectrum
and DSC analysis was unsuccessful due to protein aggregation upon higher concentrations. DLS analysis wascarried out for protein with different buffer conditions of different pH in order to determine the particle size
upon precipitation and to analyze the solubility of protein. The lowest aggregation was observed with higherhomogeneity, for MES (pH 5.5) buffer.

*Deadline: May 17, 2019
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