Purification Of Low-abundance Protein Expressed From Transgenic Plants And Its Peptide Mass Mapping

In this thesis, the method of purification and identification of Cry1Ab protein from Cry1Ab transgenic rice was developed in order to evaluate the ecological safety and environmental behavior of Cry1Ab protein expressed from Bt transgenic plants. The method of chromatographic purification and peptide mass mapping identification, as well as the protein digestion method were investigated. The method and efficiency of the trypsin immobilized by sol-gel and immobilized metal affinity chromatographic adsorbent (IMAC) were studied.In chapter one, potential ecological risks of transgenic plants, the strategy of extraction, purification and identification for the target proteins as well as the methods of immobilizing enzyme were reviewed respectively.Chapter two deals with the method of multi-column chromatography comprising ion exchange chromatography(IEC) and hydrophobic interaction (HIC) for purification of Cry1Ab protein in initial and medium steps, and high performance ion exchange column (Q-Sepharose FF) was employed in the final purification step by multi-cycles chromatographic process. Purity and apparent molecular weight of the protein was determined by SDS-PAGE. Result showed that the highly purified Cry1Ab could be obtained after the protein sample was separated by the combined chromatographic process, and high insecticidal activity remained after all the purification procedure according to the results of larvicidal test.Chapter three is aimed at identification of the purified Cry1Ab. The protein band on the gel was in-situ digested by trypsin, and the peptides mass fingerprint(PMF) was analyzed by laser desorption ionization-time of flight mass spectrometry (MALDI -TOF). The resulting peptides mass were searched on Mascot database, and 80% coverage with known Cry1Ab protein was found.Chapter four introduces a high-throughput tryptic reactor, which was prepared by coating a trypsin-containing gel on a 96-well microtiter plate. The trypsin-encapsulated gel was prepared by the sol-gel method, incorporating several siloxane reagents at various ratios. The effect of several siloxane reagents ratio on gelation speed, mechanical strength, transparency, the activity and stability of enzyme immobilized were studied respectively. Furthermore, the digestion efficiency for Bovine Serum Albumin (BSA) by immobilized enzyme and free enzyme was also compared. The peptide fragments maps were acquired by PR-HPLC. Result showed that trypsin embedded in sol-gel membrane formed by MTMS and TEOS (1:1 ratio) gave the best properties of gel, the highest enzymatic activity and higher digestion efficiency.Chapter five presents another method of immobilizing enzyme by a new immobilized metal affinity chromatography (IMAC) matrix, which was prepared by coordinating metal ion with cross-linked Chitosan coated on nonporous silica gel (M-CTS-SiO₂). Adsorption and desorption experiments were also conducted for elucidating the optimal pH, adsorption isotherm and adsorption kinetics of enzyme. Results showed that the species of chelated metal ion has a significant effect on the immobilized trypsin activity, and the maximum adsorption for trypsin by the prepared Zn-IMAC adsorbent could reach 3762±68 IU.g^-1. The results indicated that the enzyme immobilized by direct adsorption on IMAC have the advantage of easy to prepare, high enzyme activity retaining and high stability. These materials could be potentially applied in high-throughput protein digestion and peptide mass mapping.