Challenging protein purification from anammox bacteria

被引:23
作者
Cirpus, Irina E. Y.
Geerts, Wim
Hermans, John H. M.
Op den Camp, Huub J. M.
Strous, Marc
Kuenen, J. Gijs
Jetten, Mike S. M.
机构
[1] Catholic Univ Nijmegen, IWWR, Dept Microbiol, NL-6525 ED Nijmegen, Netherlands
[2] Delft Univ Technol, Dept Biotechnol, NL-2628 BC Delft, Netherlands
关键词
anammox; anaerobic ammonium oxidation; protein purification; gelation;
D O I
10.1016/j.ijbiomac.2006.02.018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:88 / 94
页数:7
相关论文
共 36 条
[1]   Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate [J].
Alting, AC ;
Hamer, RJ ;
de Kruif, GG ;
Visschers, RW .
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2000, 48 (10) :5001-5007
[2]   Effect of protein concentration, pH, lactose content and pasteurization on thermal gelation of acid caprine whey protein concentrates [J].
Bordenave-Juchereau, S ;
Almeida, B ;
Piot, JM ;
Sannier, F .
JOURNAL OF DAIRY RESEARCH, 2005, 72 (01) :34-38
[3]   A new soluble 10 kDa monoheme cytochrome c-552 from the anammox bacterium Candidatus "Kuenenia stuttgartiensis" [J].
Cirpus, IEY ;
de Been, M ;
Op den Camp, HJM ;
Strous, M ;
Le Paslier, D ;
Kuenen, GJ ;
Jetten, MSM .
FEMS MICROBIOLOGY LETTERS, 2005, 252 (02) :273-278
[4]   Anaerobic ammonium oxidation (anammox) in the marine environment [J].
Dalsgaard, T ;
Thamdrup, B ;
Canfield, DE .
RESEARCH IN MICROBIOLOGY, 2005, 156 (04) :457-464
[5]   A COLORIMETRIC METHOD FOR THE DETERMINATION OF SUGARS [J].
DUBOIS, M ;
GILLES, K ;
HAMILTON, JK ;
REBERS, PA ;
SMITH, F .
NATURE, 1951, 168 (4265) :167-167
[6]   PROTEIN GELS [J].
FERRY, JD .
ADVANCES IN PROTEIN CHEMISTRY, 1948, 4 :1-78
[7]  
Galvani M, 2001, ELECTROPHORESIS, V22, P2058, DOI 10.1002/1522-2683(200106)22:10<2058::AID-ELPS2058>3.0.CO
[8]  
2-Z
[9]   MECHANISM OF PROTEIN STABILIZATION BY GLYCEROL - PREFERENTIAL HYDRATION IN GLYCEROL-WATER MIXTURES [J].
GEKKO, K ;
TIMASHEFF, SN .
BIOCHEMISTRY, 1981, 20 (16) :4667-4676
[10]  
GRAY J, 1986, J BIOL CHEM, V261, P9282