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Titolo:
Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins
Autore:
Fox, JD; Kapust, RB; Waugh, DS;
Indirizzi:
NCI, Prot Engn Sect, Macromol Crystallog Lab, Frederick, MD 21702 USA NCIFrederick MD USA 21702 cromol Crystallog Lab, Frederick, MD 21702 USA
Titolo Testata:
PROTEIN SCIENCE
fascicolo: 3, volume: 10, anno: 2001,
pagine: 622 - 630
SICI:
0961-8368(200103)10:3<622:SAASOT>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
GREEN FLUORESCENT PROTEIN; SUBSTRATE-BINDING; IN-VIVO; RECOMBINANT PROTEINS; POLYPEPTIDE BINDING; ACTIVE-TRANSPORT; GROEL; EXPRESSION; BACTERIAL; CHAPERONE;
Keywords:
fusion protein; inclusion bodies; maltose-binding protein; protein folding; solubility; aggregation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
55
Recensione:
Indirizzi per estratti:
Indirizzo: Waugh, DS NCI, Prot Engn Sect, Macromol Crystallog Lab, POB B, Frederick, MD 21702 USA NCI POB B Frederick MD USA 21702 POB B, Frederick, MD 21702 USA
Citazione:
J.D. Fox et al., "Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins", PROTEIN SCI, 10(3), 2001, pp. 622-630

Abstract

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediatesof passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding becauseof its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6, Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

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Documento generato il 13/07/20 alle ore 07:51:02