Biomaterial Database

Simultaneous refolding and purification of a recombinant lipase with an intein tag by affinity precipitation with chitosan. 2008

Pradeep K Singh, and Munishwar N Gupta

Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India.

A strategy for simultaneous purification and refolding of proteins overexpressed with an intein tag is described. A recombinant lipase overexpressed in Escherichia coli ER2566 with the intein tag and obtained as inclusion bodies was solubilized in buffer containing 8 M urea or cetyltrimethylammonium bromide. The solubilized lipase was precipitated with chitosan and the affinity complex of the polymer with the fusion protein was obtained. The intein tag was cleaved with dithiothreitol and the refolded lipase was obtained in active form. Activity recovery of 80% was observed and the enzyme had a specific activity of 2965 units/mg. The purified lipase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purity and activity recovery were comparable with that of the preparation obtained by using the commercial kit which utilizes chromatography on chitin beads. The purified and refolded lipase was characterized by fluorescence and CD spectroscopy.

UI	MeSH Term	Description	Entries
D008049	Lipase	An enzyme of the hydrolase class that catalyzes the reaction of triacylglycerol and water to yield diacylglycerol and a fatty acid anion. It is produced by glands on the tongue and by the pancreas and initiates the digestion of dietary fats. (From Dorland, 27th ed) EC 3.1.1.3.	Triacylglycerol Lipase,Tributyrinase,Triglyceride Lipase,Acid Lipase,Acid Lipase A,Acid Lipase B,Acid Lipase I,Acid Lipase II,Exolipase,Monoester Lipase,Triacylglycerol Hydrolase,Triglyceridase,Triolean Hydrolase,Hydrolase, Triacylglycerol,Hydrolase, Triolean,Lipase A, Acid,Lipase B, Acid,Lipase I, Acid,Lipase II, Acid,Lipase, Acid,Lipase, Monoester,Lipase, Triglyceride
D011232	Chemical Precipitation	The formation of a solid in a solution as a result of a chemical reaction or the aggregation of soluble substances into complexes large enough to fall out of solution.	Precipitation, Chemical
D011993	Recombinant Fusion Proteins	Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.	Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D002686	Chitin	A linear polysaccharide of beta-1->4 linked units of ACETYLGLUCOSAMINE. It is the second most abundant biopolymer on earth, found especially in INSECTS and FUNGI. When deacetylated it is called CHITOSAN.
D002846	Chromatography, Affinity	A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D004526	Efficiency	Ratio of output to effort and or resources, or the ratio of effort and or resources produced to energy expended.	Productivity
D015202	Protein Engineering	Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.	Genetic Engineering of Proteins,Genetic Engineering, Protein,Proteins, Genetic Engineering,Engineering, Protein,Engineering, Protein Genetic,Protein Genetic Engineering
D016956	Burkholderia cepacia	A species of BURKHOLDERIA considered to be an opportunistic human pathogen. It has been associated with various types of infections of nosocomial origin.	Pseudomonas cepacia,Pseudomonas kingii,Pseudomonas multivorans
D017510	Protein Folding	Processes involved in the formation of TERTIARY PROTEIN STRUCTURE.	Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular
D047668	Inteins	The internal fragments of precursor proteins (INternal proTEINS) that are autocatalytically removed by PROTEIN SPLICING. The flanking fragments (EXTEINS) are ligated forming mature proteins. The nucleic acid sequences coding for inteins are considered to be MOBILE GENETIC ELEMENTS. Inteins are composed of self-splicing domains and an endonuclease domain which plays a role in the spread of the intein's genomic sequence. Mini-inteins are composed of the self-splicing domains only.	Introns, Protein,Protein Introns,Intervening Protein Sequence,Intein,Intervening Protein Sequences,Intron, Protein,Protein Intron,Protein Sequence, Intervening,Protein Sequences, Intervening,Sequence, Intervening Protein,Sequences, Intervening Protein