I= CHANGES IN MOLECULAR CONFORMATION OF CRYIAc PROTEIN AND RECEPTOR BINDING
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Title of Thesis
CHANGES IN MOLECULAR CONFORMATION OF CRYIAc PROTEIN AND RECEPTOR BINDING

Author(s)
Sirosh Mahmood Bokhari
Institute/University/Department Details
University of the Punjab/National centre of Excellence in Molecular Biology
Session
2002
Subject
Molecular Biology
Number of Pages
164
Keywords (Extracted from title, table of contents and abstract of thesis)
filamentous bacteriophage, protein, receptor-binding loop, cry1ac, bacillus thuringiensis, lepidopteran earias vitella

Abstract
The study presented in this thesis is endeavored to artificially evolve, the receptor-binding loop of the Cry1Ac toxin from Bacillus thuringiensis, while exploiting the filamentous bacteriophage assembly for the display and expression of mutants and their selection by Panning. The main of the research is to identify a single or few peptide motif/s, central in binding to midgut receptor proteins of lepidopteran Earias vitella.

To form the feasibility basis for affinity selection of phage-displayed Cry1Ac toxin and to study expression of its smaller fragments, various domains and the loop region of the protein were separately displayed and studied in phagemid and phage systems. The loop receptor-attachment region of native Cry1Ac from HD- 73 strain of Bt. Kurstaki, was displayed on the M13KE phage (M13KL2), in fusion with minor coat protein gIIIp. The resulting fusion-peptide of 2.2Kb, could not be detected with Cry1Ac antibodies due to absence of antigenecity determining sites in the narrow region. The expression of whole domains of Cry1Ac was investigated using a phagemid system. The sequences, encoding domain II, domains II-III and the toxin fragment, were expressed as translational fusions, with gene III in pDAN3 phagemid. It was shown that the 92 kDa domain II-gIIIp fusion peptide was most efficiently expressed although the pDH2 phage had the least titer amongst recombinants. The domain II fusion peptide also induced a toxic effect, resulting in > 90% host cell mortality. Interestingly, this property was temperature-sensitive. The 109 kDa fusion peptide of pDH23 phage was least expressed, whereas, its titer was the highest. Toxin fragment fused to the gIIIp resulted in a peptide of 139 kDa, toxic against E. vitella larvae and thus suggesting normal folding of toxin on the surface of bacteriophage.

Micropanning experiments with 125I-labeled, M13KL2 and wild-type M13KE phage using Earias vitella BBMV's as target demonstrated a selective advantage of M13KL2 over wild-type phage and thus implying its suitability for use as a target in Biopanning. Micropanning studies with pDAN3, pDH2, pDH23 and pAc phage against the receptor target, also demonstrated selectability of the recombinant phage over wild-type, however, the larger fusion peptides were subject to rapid degradation by proteolysis. Moreover, all recombinant phages exhibited some non-specific interactions of the phage coat with receptor target, as was inferred from the results of binding experiments.

Random mutagenesis of the loop II peptide sequence YRRP of Cry1Ac, followed by repeated rounds of panning against the lepidopteran receptor, did not yield any consensus binding motif. However, biopanning with a 7-mer random peptide library identified consensus binding sequence, P T/P W/T T S H F, against the E. vitella midgut receptor proteins. In vivo selection of the random peptide library in E. vitella larvae isolated the motifs, P/S N P L T/L P Q/S, which homed specifically to the receptor proteins. Alignment of the motifs, with Cry1Ac native sequence identified similar motifs at residues S394-S398 and P421-P428 in domain II, in a region established from previous studies to be important in binding and toxicity of Cry1Ac.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
105.77 KB
2 1 Introduction 1
75.06 KB
3 2 Literature Review 7
482.14 KB
  2.1 Biology Of The Filamentous Bacteriophage 7
  2.2 Changes In Native Conformation Of A Protein 23
  2.3 B acillus Thuringiensis & Its Crystal Proteins 34
4 3 Materials And Methods 43
357.29 KB
5 4 Results 70
630.95 KB
6 5 Discussion 115
213.72 KB
7 6 References 129
392.55 KB
8 7 Appendix 154
97.19 KB