Oxidative renaturation of lysozyme at high concentrations

Hevehan, Diane L.; Clark, Eliana De Bernardez

doi:10.1002/(sici)1097-0290(19970505)54:3<221::aid-bit3>3.0.co;2-h

Cited by 230 publications

(189 citation statements)

References 25 publications

Supporting

Mentioning

177

Contrasting

Unclassified

Order By: Relevance

“…As a model protein for this study, we selected hen egg-white lysozyme, whose folding has been extensively investigated (18,(23)(24)(25)(26)(27)(28). The general experimental methodology we used to examine lysozyme's folding was a classical one (18): The protein was unfolded and reduced in aqueous solution containing urea and DTT, followed by dilution into a denaturant-free solvent system containing the oxidizing mixture of oxidized and reduced glutathiones; after an incubation, the lysozyme activity was assayed in water and͞or in the solvent system itself.…”

Section: Resultsmentioning

confidence: 99%

Correct protein folding in glycerol

Rariy

Klibanov

1997

Proc. Natl. Acad. Sci. U.S.A.

137

View full text Add to dashboard Cite

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded͞reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding͞reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.The protein folding problem remains one of the key unresolved issues in biochemistry (1, 2). Specifically, despite massive research efforts and much recent progress, it is still unclear exactly how a disordered polypeptide chain spontaneously folds into a uniquely structured, biologically active protein molecule (3-6).The surrounding solvent, water, is thought to be involved inextricably in the protein folding process (7-9). Directly testing this concept experimentally would require replacing water as a medium for protein folding with a nonaqueous solvent and determining whether the protein molecule can still fold into its native conformation. This solvent replacement strategy should also yield penetrating insights into the mechanism of protein folding, as it has into the mechanisms of organic reactions (10) and enzymatic specificity (11). In addition, such an approach may shed light on intriguing observations concerning protein folding in vacuo (12)(13)(14).Studies of protein folding in nonaqueous solvents have been prevented by the common knowledge that proteins are insoluble in almost all organic solvents and that those few that do dissolve proteins are strong denaturants, such as dimethyl sulfoxide (DMSO) (15). However, our recent findings have greatly expanded the range of protein-dissolving solvents by revealing the importance of the protein having been lyophilized from aqueous solution of the ''right '' pH (16, 17). Consequently, it has become possible to address systematically the effect of the solvent on the protein folding process. As a first step toward this end, in the present work we have investigated the refolding͞reoxidation of unfolded͞reduced hen egg-white lysozyme...

show abstract

Section: Resultsmentioning

confidence: 99%

Correct protein folding in glycerol

Rariy

Klibanov

1997

Proc. Natl. Acad. Sci. U.S.A.

137

View full text Add to dashboard Cite

show abstract

“…In order to convert the inclusion body type of CADEF1 into active forms, protein refolding ensued. Since the protein would be aggregated when the denaturant was suddenly extracted away from the buffer system (Hevehan and Clark 1997), therefore, protein refolding was performed prior to protein purification. During the protein refolding, the concentration of urea was gradually decreased by dialysis, which eventually gave rise to the refolded CADEF1 with antifungal activity (Figs.…”

Section: Discussionmentioning

confidence: 99%

Antifungal activity of a recombinant defensin CADEF1 produced by Escherichia coli

2009

World J Microbiol Biotechnol

View full text Add to dashboard Cite

The defensin CADEF1 has been known to be uptranscripted in the pepper Capsicum annuum L., upon bacterial attack, abiotic elicitors and environmental stresses. However, the native form of CADEF1 has not been purified from the pepper, and its activity against fungi has been uncertain. In this study, the full-length cDNA of CADEF1 was obtained by fragment piecing-together method. Thereafter, its mature peptide coding sequence was inserted into bacterial expression vector pET28a(?), and the recombinant vector was transformed into Escherichia coli BL21 (DE3). Based on SDS-PAGE and Western blotting analysis, the recombinant CADEF1 was produced as a 5.6 kDa protein in the form of inclusion body in E. coli BL21 (DE3) and the expression level accounted for 15% of the bacterial total protein. Further processing through protein refolding and purification, converted the inclusion body type of CADEF1 into active form with the yield about 13 lg/ml of culture. The refolded CADEF1 significantly showed the activity against growth of the fungal pathogen Verticillium dahliae on average by 67.9% in vitro assay. These results verified the novel activity of CADEF1, which might benefit in prevention of the fungus-related disease in agricultural production.

show abstract

“…ARGININE was first used in refolding of human tissue type plasminogen activator [18,19]. Since then, it has been used for refolding of a variety of proteins including casein kinase II, [20], Fab antibody fragments [18,21], growth hormone [22], human gamma interferon, [23], human matrix metalloproteinase-7 human neurotrophins [24] human p53 tumor suppressor protein [25], interleukin-21 [26], interleukin-6 receptor [27], lysozyme [28], single-chain Fv fragments [29], and singlechain immunotoxins [18,30]. However, arginine may act as a protein-denaturant, which limits the expansion of its applications.…”

Section: Arginine (Arg)mentioning

confidence: 99%

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Alibolandi¹,

Mirzahosei²

2011

American J. of Biochemistry and Molecular Biology

View full text Add to dashboard Cite

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

show abstract

Oxidative renaturation of lysozyme at high concentrations

Cited by 230 publications

References 25 publications

Correct protein folding in glycerol

Correct protein folding in glycerol

Antifungal activity of a recombinant defensin CADEF1 produced by Escherichia coli

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Contact Info

Product

Resources

About