Cloning, expression, purification, and characterization of Vibrio cholerae transcriptional activator, HlyU.
The HlyU from Vibrio cholerae, involved in the transcriptional regulation of haemolysin genes, plays an important role in the regulation of virulence gene expression. We have cloned, over-expressed and purified HlyU from V. cholerae strain O395 in Escherichia coli, as an N-terminal His6-tagged protein. The purified protein gave a single band at approximately 16 kDa on SDS-PAGE, while the sequence analysis revealed the molecular weight of 15.8 kDa. The molecular mass of HlyU, determined in analytical gel-filtration experiments, was approximately 15.7 kDa, an indication that V. cholerae HlyU is a monomer. HlyU has two cysteine residues (38 and 104); reaction with sulfhydryl reagent resulted in one mol of cysteine residue reacting per mol of HlyU, while the protein denatured in guanidine hydrochloride (GdnHCl) showed the reactivity of both the residues. Circular dichroism (CD) analysis showed HlyU to be predominantly alpha-helical, while fluorescence experiment showed that the only tryptophan residue present in HlyU is solvent exposed. HlyU was found to exhibit a two-state GdnHCl-induced unfolding [DeltaG(NU)(H2O) approximately 6.2 kcal mol-1] when monitored by far-UV CD and intrinsic tryptophan fluorescence.
Saha
Rudra Prasad
RP
Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta 700 054, India.
Basu
Gautam
G
Chakrabarti
Pinak
P
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism; D008957:Models, Genetic; D017510:Protein Folding; D012997:Solvents / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D012997:Solvents; D015534:Trans-Activators; D014157:Transcription Factors; C084286:hlyU protein, Vibrio cholerae; D004228:Dithionitrobenzoic Acid; D019791:Guanidine; D003545:Cysteine