Challenging protein purification from anammox bacteria.
The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.
Cirpus
Irina E Y
IE
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Geerts
Wim
W
Hermans
John H M
JH
Op den Camp
Huub J M
HJ
Strous
Marc
M
Kuenen
J Gijs
JG
Jetten
Mike S M
MS
D000641:Ammonia / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000737:chemistry* / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c / Q000302:isolation & purification* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D000641:Ammonia; D045304:Cytochromes c