Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenulatus.
The His-tagged lipase BTL2 from Bacillus thermocatenulatus was expressed in Escherichia coli and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography. The success of protein separation and purification was pH-dependent and increased with decreasing pH. The purified BTL2 lipase showed a strong tendency to aggregate upon concentration, which prevented a reproducible crystallization. Aggregation studies using dynamic light-scattering (DLS) analysis were performed to improve the purification and concentration of BTL2 lipase. Different chemical classes of additives were tested to manipulate the aggregation behaviour of BTL2 lipase with the aim of obtaining a monodisperse sample to use for crystallization. For the process of concentration of BTL2 lipase in monomeric form, the alcohol 2-propanol and the ionic detergent dodecyl dimethylamine-N-oxide (LDAO) were found to be necessary. For the concentrated lipase, the availability of 5% 2-propanol was sufficient to hold the lipase in monomeric form and no additional detergent was needed.
Schlieben
Nils Helge
NH
Universität zu Köln, Institut für Biochemie, Zülpicher Strasse 47, 50674 Köln, Germany.
Niefind
Karsten
K
Schomburg
Dietmar
D
D019840:2-Propanol / Q000737:chemistry / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D003902:Detergents / Q000494:pharmacology; D004123:Dimethylamines / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005990:Glycerol / Q000737:chemistry / Q000494:pharmacology; D006018:Glycols / Q000737:chemistry / Q000494:pharmacology; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D010168:Palmitates / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D012995:Solubility / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D003902:Detergents; D004123:Dimethylamines; D006018:Glycols; D010168:Palmitates; D011993:Recombinant Fusion Proteins; C050015:4-nitrophenyl palmitate; D011092:Polyethylene Glycols; C014518:dodecyldimethylamine oxide; D006639:Histidine; D008049:Lipase; D019840:2-Propanol; D005990:Glycerol