Peptides (eBook)

Preface 5
Contents 11
Abbreviations 19
Session A Novel approach on synthetic method 21
Orthogonal ligation of free peptides 22
Solid phase synthesis of peptide aldehydes 27
Investigation of enzyme activity and inhibition in the interior of novel solid supports 33
Immobilization of a-Chymotrypsin on zeolite for peptide synthesis in organic solvent 40
Preparation of a peptide thioester using a fluoren-9-ylmethoxycarbonyl solid-phase method 45
Application of DEPBT for the synthesis of N-protected peptide alcohols 49
Decomposition of amino acid cupric complex by using tetrahydrothiazole-2-thione for preparation ofN a-Boc-Ne-Fmoc-L-Lysine 52
Two-step selective formation of three disulfide bridges in the synthesis of d-Gonotoxin PVIA 55
Session B Structure, folding and conformation analysis De novo design of peptide & protein
Design, synthesis and NMR structure of a rigid cyclic peptide template 60
Automated docking of Sch68631 with HCV NS3 protease 65
Molecular modeling of the three-dimensional structure of the human interleukin-11 68
Designing ß-hairpin forming short peptide 72
Anti-tumor activities of papavor somniferum pollen peptides 76
Structural investigation of two pollen peptides by 2 D homonuclear NMR spectroscopy 79
A comparison of three heuristic algorithms for molecular docking 82
Aggregation of a calmodulin-binding peptide 86
Session C Neuro/Endocrino/Bioactive peptide 90
Neuropeptide AVP(4-8) playing an indirect neurotrophic role 91
Potent bradykinin antagonists having medical potential 93
Understanding the chemistry and biology of rodent relaxin: Role of the C-peptide 97
Purification and characterization of a new conotoxin from the venom ofConus betulinus 102
Synthesis and biological activity of human calcitonin analogs 105
Enhanced expression of CNDF mRNA in rat brain by administration of AVP(4-8) 108
The synthesis and opioid activities of nociceptin and its fragments 111
The effect of Glu position change on the opioid activity of deltorphin II 114
Isolation and purification of a series of peptides from Panax notoginseng 118
Study of the relationship between structure and function of HIV-1 gp 41 N terminus fusion peptide 120
Hypotensive activities of nociceptin and its fragments 124
The design and synthesis of endomorphins and their analogues 128
Neuronal apoptosis induced by ß-amyloid peptides in vitro 132
Studies on the spin-labeling technique on deltorphin II and its analogues 135
Design and synthesis of salmon calcitonin and its analogues 140
Studies on the synthesis and antithrombosis activity of two peptides derived from the molecular-binding site of fibrinogen 142
Synthesis and expression of w-conotoxin MVIIA 145
Session D Peptide vaccines/Immunology/Virus 150
Studies on the synthetic peptide vaccines against schistosomiasis 151
Studies on the anti-schistosomal synthetic peptide vaccine: Prediction and synthesis of antigenic peptides of Schistosoma paramyosin 154
Construction and expression of a recombinant antibody targeted plasminogen activator 156
Chemical conjugation between haemophilus influenzae type b (hib) polysaccharide and proteins 159
Synthesis of human inhibin fragments, preparation and generation of monoclonal antibodies against human inhibin 161
Session E Peptide diversity Chemical libraries
Design of a d opioid peptide mimetic based on a topographically constrained cyclic enkephalin 166
Tetrazole cis-amide bond mimetics identify the ß-turn conformation of insect kinin neuropeptides 171
Synthetic studies of farnesyl protein transferase inhibitor pepticinnamin E 176
Novel coupling reagents 179
Pharmacophore model of growth hormone secretagogues 182
Discovery that deltorphin II derivatives are potent melanotropins, putatively active at the Xenopus melanocortin- 1 receptor 185
Session F Glyco/Lipo/Phospho peptide Peptide diversity/Chemical libraries
Enzymatic synthesis of phosphopeptides 189
Synthesis of phosphorylated polypeptide by a thioester method 192
Difference between N-phospho-L- and D-methionine in ester exchange reaction with uridine 198
Synthesis of analogs of phosphoamino acids and their biomimic reactions 200
Solid phase synthesis of heterocyclic combinatorial libraries derived from peptides 204
Parallel synthesis of muramyl peptides derivatives 210
ß-Cyclodextrin for presentation of bioactive peptides to molecular recognition 214
The C-terminus of d-opioid receptor does not control DPDPEinduced desensitization 222
Session G Peptides in therapeutics and diagnosis 225
Detection of gene expression product of transgenic tobacco by antigenic peptide 226
Advantages of Calcitonin complexed with SA liposome 228
Hypoglycemic effects of insulin sublingual drops containing azone on rats and rabbits 232
Session H Other research related to peptides and proteins 235
Secretory expression of porcine insulin precursor in methylotrophic yeast Pichia pastoris 236
Chemo-enzymatic syntheses of eel calcitonin analogs having natural N-linked oligosaccharides 239
Design of specific inhibitors at the ATP binding site of DNA topoisomerases II 243
Applications of optical biosensors to structure-function studies in the EGF/EGF receptor system 247
Experimental study of osteogenic growth peptide promoting bone mass in rat osteoporosis 253
Determination of inhibitory constants for CPA by competitive spectrophotometry 256
Sublingual administration of monomeric insulin - destetrapeptide insulin 258
How to get patent rights to protect your invention in biotechnology 261
Study on peptide maps from milk protein hydrolysates by SEHPLC 264
Synthesis and solution structures of new chiral enantiomeric peptide nucleic acid (PNA) dimers 267
Study of one type of metallothionein and SZ51 recombinant protein directing and imaging reagent 271
LHRH antagonists: new preclinical and clinical results 273
DNA-binding characteristics of antitumor drug actinomycin D and its analogs 278
Design, synthesis of the peptide analogs of antitumor agent actinomycin D 283
Authors Index 288
Subject Index 291

Detection of gene expression product of transgenic tobacco by antigenic peptide (p. 215-216)

Jia-Xi Xu a*, Yuan Ma b, Mi Ma c and Zhong-Ping Lin d
aDepartment of Chemistry, Peking University. Beijing, 100871,
bDepartment of Chemistry, Tsinghua University, Beijing, 100084,
cInstitute of Botany, Academia Sinica, Beijing 10044,
dDepartment of Biology, Peking University, Beijing, 100871, China

Introduction

The need as well as the ability to cultivate special plants, such as antiviral wheat, antiinsect plants, economical plants etc, has increased with advances in biological science and technology and has created significant economic benefits. It is easy to detect DNA and RNA in transgenic processing. However, it is difficult to detect the protein as gene expression product due to low concentration and difficult separation and purification (1). Now we have used a synthetic antigenic peptide to solve this problem. After predicting and synthesizing an antigenic peptide of isopentyl transferase in transgenic tobacco, a key enzyme in gene expression, a peptide with the sequence IHARQQEQKF was conjugated to BSA. Its antibodies were then obtained from rabbit sera after immunizing the rabbit with this conjugated antigen. The antibody can be used for the qualitative and quantitative determination of gene expression product in crude proteinextract of leaf, stem and root of transgenic tobacco by ELISA and Western blot methods.

Results and Discussion
Prediction of epitope

The nucleotide and amino acid sequences of the isopentyl transferase in transgenic tobacco was obtained from published data (2). Its epitope was predicted according to the hydrophilicity (Hopp and Woods method), flexibility and accessibility by the computer program PC-Gene (3,4). Its secondary structure was predicted by the Chou and Fasman method (5). Peptide IHARQQEQKF (212-221 amino acid residues) with high hydrophilicity, flexibility and accessibility was predicted.

Synthesis of epitopic peptides

The predicted peptide was synthesized by Merrifield solid phase peptide synthesis method with the acid-labile tert-butyloxycarbonyl (Boc) group for temporary protection and acid-stable groups for side chain protection (3, 4, 6). Side chain-protected peptideresin was cleaved by anhydrous hydrogen fluoride under anisole, 1, 2-ethandithiol and thioanisole as scavengers and washed with cooled ethyl ether. Peptide was extracted with 30% acetic acid and purified by gel filtration on Sephadex G10. It was further purified by preparative HPLC and checked for homogeneity by reverse-phase HPLC. Its structure was confirmed by amino acid analysis and FAB-MS.

Immunologiccal methods

This antigenic peptide was conjugated to BSA by the glutaraldehyde method and its antibodies were then obtained from rabbit sera after immunizing rabbit with this conjugated antigen (7). The antibody was used to detect the gene expression product of transgenic tobaco at a dilution of 1:132 with OD492 nm 0.95 by the ELISA method. The results are shown in Table 1. This is a simple method for the qualitative and quantitative determination of gene expression product in crude protein-extract of leaf, stem and root of transgenic tobacco using ELISA and Western blot methods.