Cyclodextrins as Protein Folding Aids

Karuppiah, N.; Sharma, Ajit

doi:10.1006/bbrc.1995.1778

Cited by 126 publications

(109 citation statements)

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“…After our original description of the artificial chaperone technique appeared (21), Karuppiah and Sharma (41) reported that cyclodextrin alone, used as a dilution additive, could promote CAB refolding from the Gdm-denatured state. Most of the refolding conditions employed by these workers, however, allowed substantial reactivation yields in the absence of cyclodextrin.…”

Section: Comparison Of the Artificial Chaperone Methods With Other Refmentioning

confidence: 99%

Artificial Chaperone-assisted Refolding of Carbonic Anhydrase B

Rozema¹,

Gellman²

1996

Journal of Biological Chemistry

198

163

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We recently reported a new approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and a cyclodextrin (Rozema, D., and Gellman, S. H. (1995) J. Am. Chem. Soc. 117, 2373-2374). Here, we provide a detailed study of carbonic anhydrase B (CAB) refolding assisted by these "artificial chaperones." When CAB is heated in the presence of a competent detergent, or when guanidiniumdenatured CAB is diluted to nondenaturing guanidinium concentration in the presence of such a detergent, the detergent forms a complex with the nonnative protein, thereby preventing aggregation. CAB is unable to refold from the detergent-complexed state, but folding can be induced by introduction of a cyclodextrin, which strips the detergent away from the protein.Use of artificial chaperones provides excellent yields of reactivated CAB under conditions that lead to little or no reactivation in the absence of the refolding assistants. Our studies show that the detergent can capture the unfolded protein even at submicellar concentrations, but that not all CAB-detergent complexes lead efficiently to refolded enzyme upon introduction of the stripping agent. Effective refolding appears to require that detergent stripping occur as rapidly as possible; intrinsically slow methods of detergent removal (dialysis or use of macroscopic adsorbents) are less effective than cyclodextrin at inducing renaturation upon detergent removal. The detailed characterization of artificial chaperone-assisted CAB refolding reported here should guide the application of this strategy to other proteins.The process by which protein molecules achieve their native conformations is a subject of fundamental and practical importance. Fundamental interest in the "protein folding problem" arises because we do not yet understand how a complex network of noncovalent interactions can specify one particular compact conformation for an intrinsically flexible polypeptide (1), how the polypeptide rapidly finds that compact conformation (2, 3), or why such a purely noncovalent process involves relatively large kinetic barriers (4). Practical interest in the "protein refolding problem" stems from the fact that proteins overproduced by genetically engineered cells are often obtained in non-native forms (e.g. inclusion bodies), and the use of such proteins for basic research or biotechnological applications requires that the native conformation be achieved (5, 6).Revolutionary advances in genetic manipulation techniques have made protein refolding an increasingly pressing problem, but there have been relatively few efforts to devise general renaturation strategies. In a common scenario, an overproduced protein is purified in inclusion body form, the inclusion bodies are solubilized with a chemical denaturant (typically guanidinium or urea), and refolding is attempted by removing the denaturant, via dilution or dialysis. For many proteins, however, denaturant removal leads predominantly or completely to protein aggregation rather than refolding (...

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Section: Comparison Of the Artificial Chaperone Methods With Other Refmentioning

confidence: 99%

Artificial Chaperone-assisted Refolding of Carbonic Anhydrase B

Rozema¹,

Gellman²

1996

Journal of Biological Chemistry

198

163

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“…In general, these compounds produced an inhibition of aggregation higher than 50% at concentration above 15% w/v (Table 1). Although cyclodextrins have been proposed as folding aids and inhibitors of protein aggregation, 29 they do not appear to inhibit the aggregation of RSV (Fig. 1A and Table 1).…”

Section: High Throughput Screening For Inhibitors Of Rsv Aggregationmentioning

confidence: 99%

High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

Ausar

Espina²,

Brock³

et al. 2007

Human Vaccines

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Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in children worldwide. Recombinant live attenuated viral preparations are one of the most promising strategies for vaccination but they typically possess poor thermostability. In this work, a library of compounds was screened and stabilizers were selected based on their ability to inhibit the aggregation of RSV perturbed at 56˚C. After screening and selection of excipients, the conformational stability of the RSV proteins was evaluated in the presence of potential stabilizers. The secondary and tertiary structures as well as aggregation/dissociation of RSV were monitored using circular dichroism and second derivative UV absorption spectroscopies and light scattering, respectively, as a function of temperature (10-90˚C). RSV membrane fluidity was also evaluated by generalized polarization of Laurdan fluorescence. Screening experiments showed that a variety of sugars, amino acids, polyols and polyanions inhibited the aggregation of viral particles. Conformational stability studies demonstrated that the addition of sugars and polyols stabilized RSV as indicated by a significant increase in the transition melting temperature (Tm) of both the secondary and tertiary structures as well as the gel to liquid crystalline membrane transition. These results should provide the basis for rational development of more physically stable formulations of live attenuated RSV vaccines.

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“…CAB preferred ionic detergent as the ¢rst agent (Table 2). Cetyltrimethylammonium bromide (CTAB) and N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB [3][4][5][6][7][8][9][10][11][12][13][14] were the most e¡ective detergents. Fortunately, cycloamylose also worked well in stripping these ionic detergents and promoting refolding of CAB at a high yield.…”

Section: Refolding Of Cab and Lysozymementioning

confidence: 99%

“…One common approach, known as the`dilution additive strategy', has been to include low molecular weight folding assistants in the bu¡er used to dilute the chemically denatured protein. A number of in vitro aggregation inhibitors or folding aids such as polyethylene glycol [4], polyamino acids [5], cyclodextrins [6] and detergents [7] have been reported to prevent aggregation and enhance protein folding.…”

Section: Introductionmentioning

confidence: 99%

Cycloamylose as an efficient artificial chaperone for protein refolding

et al. 2000

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High molecular weight cyclic K K-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. ß

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Cyclodextrins as Protein Folding Aids

Cited by 126 publications

References 0 publications

Artificial Chaperone-assisted Refolding of Carbonic Anhydrase B

Artificial Chaperone-assisted Refolding of Carbonic Anhydrase B

High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

Cycloamylose as an efficient artificial chaperone for protein refolding

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