Selective Extraction Of Periplasmic Proteins From Acidithiobacillus Ferrooxidans And Establishment Of The Differential Two-Dimensional Electrophoresis Maps

Acidithiobacillus ferrooxidans ( A. ferrooxidans ) is a chemolithotrophic bacterium that obtains its energy from the oxidation of ferrous iron, elemental sulfur or partially oxidized sulfur compounds. At present, A. ferrooxidans is the most employed and lucubrated bacteria in bioleaching. The process A. ferrooxidans obtaining its energy is very complicated, most of energy oxidation occurred in periplasmic space of the cell, data about sulfur oxidation pathway is very limited, in order to make sure key proteins function in sulfur oxidation pathway, it is much more efficient to focus on the proteins in periplasmic space than the whole proteins in cell.In this paper, the periplasmic proteins extraction methods have been compared in terms of the yield of proteins and MDH activity and determined that osmotic shock method is the most efficient method to extract the periplasmic proteins of A. ferrooxidans. The osmotic shock method has been modified to extract periplasmic proteins as many as possible meanwhile make sure the contamination of cytoplasmic proteins as low as possible, then this modified method has been verified. The modified results are: to extract 0.1 g (wet weight)cell, the extracting time is 15 min, the molar ratios of hyper- to hypo-osmotic pressure is 1:1, the osmotic solvent volume is 10 mL.Experiment of determination of proper quantity of 2-DE sample, voltage of IEF and electric current in SDS-PAGE are carried out. The quantity of 2-DE sample is 300μg, voltage of IEF programmed as 30V ,12 h; 500 V ,5 h; 1000 V ,1 h; 3000 V ,30 min; 5000 V ,30 min; 7000V ,1 h; 8000 V ,6 h. Electric current in SDS-PAGE programmed as 10 mA, 60 min; 25mA, 7 h.By the modified osmotic shock method, the periplasmic proteins in different growth periods of A. ferrooxidans ATCC23270 were prepared and performed 2-DE, the difference in these electrophoregrams were comparatively analyzed in terms of the cellular physiological and morphological characteristics. And we discovered: Periplasmic proteins in exponential and stadium phase are most with minor molecular weight and alkaline. Some spots in exponential phase gel are disappeared in stadium phase gel, some spots with larger molecular weight have appeared; As cells apoptosis in dead phase,there are obviously contamination with cytoplasmic proteins in periplasmic protein extraction solution, and the spots are getting more in dead phase gel. These results could be useful in key redox proteins selection by differential expression analysis in the future.