[Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein].
OBJECTIVE
To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae.


METHODS
Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis.


RESULTS
The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths.


CONCLUSIONS
The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.
Li
Wan-yi
WY
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. hxliwany@126.com
Kuang
Yu
Y
Li
Ming-yuan
MY
Yang
Yuan
Y
Jiang
Zhong-hua
ZH
Yao
Feng
F
Chen
Chang-chun
CC
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002021:Buffers; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration
D001426:Bacterial Proteins; D002021:Buffers; C035832:hook protein, bacterial flagellum