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Engineered antibodies: Folding stabi...

Tan, Philip H.

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Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility./
Author:	Tan, Philip H.
Description:	176 p.
Notes:	Source: Dissertation Abstracts International, Volume: 58-08, Section: B, page: 4205.
Contained By:	Dissertation Abstracts International58-08B.
Subject:	Chemistry, Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9807035
ISBN:	0591572389

Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility.
Tan, Philip H.

Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility. - 176 p.

Source: Dissertation Abstracts International, Volume: 58-08, Section: B, page: 4205.

Thesis (Ph.D.)--University of Washington, 1997.

In this research, we have successfully constructed and expressed 2 different single-chain Fvs. The S5 scFv is directed against the CD44 antigen and the A6H scFv is directed against a renal cell carcinoma cell receptor marker. The proteins were expressed in inclusion bodies in Escherichia coli using the T7 expression system and refolded to the native state employing refolding buffer consisting of L-Arginine and the redox system of Cystiene/Cystine pair. S5 scFv retained complete activity comparable to its Fab' fragment whereas A6H scFv retained good but not complete activity.

ISBN: 0591572389Subjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility.
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020 $a 0591572389
035 $a (UnM)AAI9807035
035 $a AAI9807035
040 $a UnM $c UnM
100 1 $a Tan, Philip H. $3 1929044
245 1 0 $a Engineered antibodies: Folding stability, domain-domain assembly, refolding efficiency and solubility.
300 $a 176 p.
500 $a Source: Dissertation Abstracts International, Volume: 58-08, Section: B, page: 4205.
500 $a Chairperson: Patrick S. Stayton.
502 $a Thesis (Ph.D.)--University of Washington, 1997.
520 $a In this research, we have successfully constructed and expressed 2 different single-chain Fvs. The S5 scFv is directed against the CD44 antigen and the A6H scFv is directed against a renal cell carcinoma cell receptor marker. The proteins were expressed in inclusion bodies in Escherichia coli using the T7 expression system and refolded to the native state employing refolding buffer consisting of L-Arginine and the redox system of Cystiene/Cystine pair. S5 scFv retained complete activity comparable to its Fab' fragment whereas A6H scFv retained good but not complete activity.
520 $a We have examined the role of the V $\ sb{\rm H} $- V $\ sb{\rm L} $ interface in scFv thermodynamic folding stability, refolding efficiency and domain-domain assembly by performing site-directed mutagenesis studies on the highly conserved hydrogen bonding between two glutamates across the V $\ sb{\rm H} $- V $\ sb{\rm L} $ interface. We showed that stability of S5 scFv can be significantly improved when double mutations consisting of methionine and alanine or glutamate and lysine were made to replace the two glutamates. The increase in stability was likely due to an increase in V $\ sb{\rm H} $- V $\ sb{\rm L} $ interaction free energy and/or destabilization of the unfolded state. We also found some of these mutations, particularly the single or double methionines mutations resulted in significant increase in refolding efficiency and suppressed aggregation. We also made thermodynamic studies of the isolated V $\ rm\sb{H},V\sb{L} $ and Fv antibody derivatives and provided insight into the individual contribution of these units to folding stability. Our finding showed that scFv linker is destabilizing to our model scFv antibody by preventing optimal V $\ sb{\rm H} $- V $\ sb{\rm L} $ interactions and/or affecting the domain folding. Using the A6H system as a model, we showed that scFv solubility can be improved by engineering its pI through addition of a stretch of glutamic acid residues at the C-terminus.
520 $a The research may provide technological routes to improving protein stability, refolding efficiency and solubility, giving an opportunity for protein engineer to introduce desirable characteristics into single-chain Fv for medical and biotechnology applications.
590 $a School code: 0250.
650 4 $a Chemistry, Biochemistry. $3 1017722
650 4 $a Biophysics, Medical. $3 1017681
650 4 $a Engineering, Biomedical. $3 1017684
690 $a 0487
690 $a 0760
690 $a 0541
710 2 0 $a University of Washington. $3 545923
773 0 $t Dissertation Abstracts International $g 58-08B.
790 1 0 $a Stayton, Patrick S., $e advisor
790 $a 0250
791 $a Ph.D.
792 $a 1997
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9807035