Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803.
The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.
Schwabe
Tatjana M E
TM
Plant Biochemistry, Faculty of Biology, Ruhr-University Bochum, Universitaetsstr. 150, ND 3/130, 44801, Bochum, Germany. tatjana.schwabe@ruhr-uni-bochum.de
Gloddek
Kirsten
K
Schluesener
Daniela
D
Kruip
Jochen
J
D001426:Bacterial Proteins; D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000737:chemistry*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D045331:Photosystem I Protein Complex / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; C105031:BtpA protein, Synechocystis sp. PCC 6803; D017931:DNA Primers; D008565:Membrane Proteins; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins; C109165:Ycf3 protein, bacteria