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Titolo:
Phage display selection of hairpin loop soyacystatin variants that mediatehigh affinity inhibition of a cysteine proteinase
Autore:
Koiwa, H; DUrzo, MP; Assfalg-Machleidt, I; Zhu-Salzman, K; Shade, RE; An, HJ; Murdock, LL; Machleidt, W; Bressan, RA; Hasegawa, PM;
Indirizzi:
Purdue Univ, Ctr Plant Environm Stress Physiol, W Lafayette, IN 47907 USA Purdue Univ W Lafayette IN USA 47907 s Physiol, W Lafayette, IN 47907 USA LMU Muenchen, Inst Physiol Chem Phys Biochem & Zellbiol, D-80336 Munich, Germany LMU Muenchen Munich Germany D-80336 & Zellbiol, D-80336 Munich, Germany Texas A&M Univ, Dept Entomol, College Stn, TX 77843 USA Texas A&M Univ College Stn TX USA 77843 ntomol, College Stn, TX 77843 USA Purdue Univ, Dept Entomol, W Lafayette, IN 47907 USA Purdue Univ W Lafayette IN USA 47907 t Entomol, W Lafayette, IN 47907 USA Auburn Univ, Dept Nutr & Food Sci, Auburn, AL 36849 USA Auburn Univ Auburn AL USA 36849 ept Nutr & Food Sci, Auburn, AL 36849 USA
Titolo Testata:
PLANT JOURNAL
fascicolo: 5, volume: 27, anno: 2001,
pagine: 383 - 391
SICI:
0960-7412(200109)27:5<383:PDSOHL>2.0.ZU;2-B
Fonte:
ISI
Lingua:
ENG
Soggetto:
COLORADO POTATO BEETLE; RAY CRYSTAL-STRUCTURE; EGG-WHITE CYSTATIN; CHICKEN CYSTATIN; CATHEPSIN-B; METHYL JASMONATE; PROTEASE INHIBITORS; MOLECULAR-CLONING; ESCHERICHIA-COLI; ORYZACYSTATIN-I;
Keywords:
proteinase inhibitor; cystatin; phage display; cysteine proteinase; molecular evolution;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
63
Recensione:
Indirizzi per estratti:
Indirizzo: Hasegawa, PM Purdue Univ, Ctr Plant Environm Stress Physiol, W Lafayette, IN 47907 USA Purdue Univ W Lafayette IN USA 47907 Lafayette, IN 47907 USA
Citazione:
H. Koiwa et al., "Phage display selection of hairpin loop soyacystatin variants that mediatehigh affinity inhibition of a cysteine proteinase", PLANT J, 27(5), 2001, pp. 383-391

Abstract

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 10(10)-10(11) phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower K-i (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 X 10(7) M(-1)sec(-1)). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

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Documento generato il 07/07/20 alle ore 12:57:52