PURIFICATION OF HYDROPHOBIC INTEGRAL MEMBRANE-PROTEINS FROM MYCOPLASMA-HYOPNEUMONIAE BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A general and practical approach for isolating, fractionating and puri fying large quantities of outer membrane hydrophobic proteins is descr ibed as applied to membrane proteins of Mycoplasma hyopneumoniae. Oute r membrane proteins were extracted with Triton X-114 detergent and wer e precipitated from the detergent phase with 90% ethanol. Precipitated proteins were dissolved in 65% formic acid and separated by RP-HPLC u sing a formic acid-acetonitrile gradient. A M(r) 48 000 protein was ob tained in high yield and at greater than 90% purity by optimisation of parameters for RP-HPLC. The combination of Triton X-114 extraction fo llowed by high resolution RP-HPLC is a novel and rapid procedure for t he isolation and purification of hydrophobic proteins. Proteins purifi ed by this approach were suitable for subsequent characterisation by d irect sequencing of the amino terminus as well as generation of peptid es by digestion with cyanogen bromide.