DIRECTED EVOLUTION OF A SUBTILISIN WITH CALCIUM-INDEPENDENT STABILITY

被引：87

作者：

STRAUSBERG, SL

ALEXANDER, PA

GALLAGHER, DT

GILLILAND, GL

BARNETT, BL

BRYAN, PN

机构：

[1] UNIV MARYLAND,MARYLAND BIOTECHNOL INST,CTR ADV RES BIOTECHNOL,ROCKVILLE,MD 20850

[2] NATL INST STAND & TECHNOL,ROCKVILLE,MD 20850

[3] PROCTER & GAMBLE CO,MIAMI VALLEY LABS,CINCINNATI,OH 45239

来源：

BIO-TECHNOLOGY | 1995年 / 13卷 / 07期

关键词：

D O I：

10.1038/nbt0795-669

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Extracellular proteases of the subtilisin-class depend upon calcium for stability. Calcium binding stabilizes these proteins in natural extracellular environments, but is an Achilles' heel in industrial environments which contain high concentrations of metal chelators. Here we direct the volution of calcium-independent stability in subtilisin BPN'. By deleting the calcium binding loop from subtilisin, we initially destabilize the protein but create the potential to use new structural solutions for stabilization. Analysis of the structure and stability of the loop-deleted prototype followed by directed mutagenesis and selection for increased stability resulted in a subtilisin mutant with native-like proteolytic activity but 1000-times greater stability in strongly chelating conditions.

引用

收藏

页码：669 / 673

页数：5

相关论文

共 13 条

[1] CRYSTAL-STRUCTURE OF THE ALKALINE PROTEINASE SAVINASE FROM BACILLUS-LENTUS AT 1.4-A RESOLUTION [J].

BETZEL, C ;

KLUPSCH, S ;

PAPENDORF, G ;

HASTRUP, S ;

BRANNER, S ;

WILSON, KS .

JOURNAL OF MOLECULAR BIOLOGY, 1992, 223 (02) :427-445

[2] THE HIGH-RESOLUTION X-RAY CRYSTAL-STRUCTURE OF THE COMPLEX FORMED BETWEEN SUBTILISIN CARLSBERG AND EGLIN-C, AN ELASTASE INHIBITOR FROM THE LEECH HIRUDO-MEDICINALIS - STRUCTURAL-ANALYSIS, SUBTILISIN STRUCTURE AND INTERFACE GEOMETRY .2. [J].

BODE, W ;

PAPAMOKOS, E ;

MUSIL, D .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1987, 166 (03) :673-692

[3] SITE-DIRECTED MUTAGENESIS AND THE ROLE OF THE OXYANION HOLE IN SUBTILISIN [J].

BRYAN, P ;

PANTOLIANO, MW ;

QUILL, SG ;

HSIAO, HY ;

POULOS, T .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (11) :3743-3745

[4] ENERGETICS OF FOLDING SUBTILISIN BPN' [J].

BRYAN, P ;

ALEXANDER, P ;

STRAUSBERG, S ;

SCHWARZ, F ;

LAN, W ;

GILLILAND, G ;

GALLAGHER, DT .

BIOCHEMISTRY, 1992, 31 (21) :4937-4945

[5]

BRYAN P N, 1986, Proteins Structure Function and Genetics, V1, P326

[6] CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN [J].

GALLAGHER, T ;

BRYAN, P ;

GILLILAND, GL .

PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1993, 16 (02) :205-213

[7]

GROS P, 1991, J BIOL CHEM, V266, P2953

[8] STRUCTURAL COMPARISON OF 2 SERINE PROTEINASE PROTEIN INHIBITOR COMPLEXES - EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO [J].

MCPHALEN, CA ;

JAMES, MNG .

BIOCHEMISTRY, 1988, 27 (17) :6582-6598

[9] LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN' THROUGH INCREMENTAL CHANGES IN THE FREE-ENERGY OF UNFOLDING [J].

PANTOLIANO, MW ;

WHITLOW, M ;

WOOD, JF ;

DODD, SW ;

HARDMAN, KD ;

ROLLENCE, ML ;

BRYAN, PN .

BIOCHEMISTRY, 1989, 28 (18) :7205-7213

[10] THE ENGINEERING OF BINDING-AFFINITY AT METAL-ION BINDING-SITES FOR THE STABILIZATION OF PROTEINS - SUBTILISIN AS A TEST CASE [J].

PANTOLIANO, MW ;

WHITLOW, M ;

WOOD, JF ;

ROLLENCE, ML ;

FINZEL, BC ;

GILLILAND, GL ;

POULOS, TL ;

BRYAN, PN .

BIOCHEMISTRY, 1988, 27 (22) :8311-8317