Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new two-step procedure of protein refolding in vitro, proposed by Rozema and Gellman and named artificial chaperone-assisted refolding, is discussed. The new approach has been inspired by the two-step mechanism of the GroE system. In the first step, the protein is captured by a detergent under conditions that would normally lead to irreversible protein aggregation (heating or denaturant removal). In the second step, removal of detergent from the protein--detergent complex is triggered by addition of a cyclodextrin which is capable of forming "inclusion complexes" with detergent, allowing the protein to refold. The protein refolded with artificial chaperones (detergent and cyclodextrin) may be purified via a two-step protocol. After refolding was complete, the solution was passed through a 0. 22-micro(m) filter, to remove aggregated protein, and then through a M = 10 kD cutoff filter. The second filtration was intended to allow the low-molecular-weight artificial chaperones to pass, but to retain the refolded enzyme. The application of the above procedure for refolding of carbonic anhydrase B from human erythrocytes, hen egg white lysozyme, pig heart citrate synthase, and creatine kinase from rabbit skeletal muscles (MM isoenzyme) is discussed.
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PMID:Artificial chaperone-assisted refolding of proteins. 955 24

The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.
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PMID:Artificial chaperone-assisted refolding of citrate synthase. 985 49

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.
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PMID:Cycloamylose as an efficient artificial chaperone for protein refolding. 1111 53