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 purifying large quantities of outer membrane hydrophobic proteins is described as applied to membrane proteins of Mycoplasma hyopneumoniae. Outer membrane proteins were extracted with Triton X-114 detergent and were precipitated from the detergent phase with 90% ethanol. Precipitated proteins were dissolved in 65% formic acid and separated by RP-HPLC using a formic acid-acetonitrile gradient. A M(r) 48 000 protein was obtained in high yield and at greater than 90% purity by optimisation of parameters for RP-HPLC. The combination of Triton X-114 extraction followed by high resolution RP-HPLC is a novel and rapid procedure for the isolation and purification of hydrophobic proteins. Proteins purified by this approach were suitable for subsequent characterisation by direct sequencing of the amino terminus as well as generation of peptides by digestion with cyanogen bromide.
Lee
R P
RP
Department of Parasitology, University of Queensland, St. Lucia, Australia.
Doughty
S W
SW
Ashman
K
K
Walker
J
J
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009174:Mycoplasma / Q000737:chemistry*; D017830:Octoxynol; D011092:Polyethylene Glycols
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40