Expression of Exogenous Gene in Prokaryotes and Eukaryotes

[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.

Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase（BChE, EC 3.1.1.8）, which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene（h BCh E） as published. The ratio of guanine and cytosine in four kinds of bases（（G＋C） ratio） was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range（〉60% activity at pH 4.0 to 8.0）. Moreover, it had a good adaptability to high temperature（〉60% activity at both 50 and 60°C up to 60 min） and good stability at 70°C. The enzyme can be activated by Li^＋, Co^＋, Zn^2＋ and ethylene diamine tetraacetic acid（EDTA）, but inhibited by Mg^2＋, Mn^2＋, Fe^2＋, Ag^＋ and Ca^2＋. Na^＋ had little effect on its activity. The values of h BChE of the Michaelis constant（Km） and maximum reaction velocity（Vm） were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants（K_i） of the hBChE to four pesticides were similar with that of electric eel AChE（EeAChE） and higher than that of horse BChE（HoBChE）. All vlues of the half maximal inhibitory concentration of a substance（IC50） for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.

An evaluation of garlic lectin as an alternative carrier domain for insecticidal fusion proteins

The mannose-binding lectin GNA （snowdrop lectin） is used as a＂carrier＂ domain in insecticidal fusion proteins which cross the insect gut after oral ingestion. A similar lectin from garlic bulb, ASAII, has been evaluated as an alternative ＂carrier＂. Recombinant ASAII delivered orally to larvae of cabbage moth （Mamestra brassica; Lepidoptera） was subsequently detected in haemolymph, demonstrating transport. Fusion proteins comprising an insect neurotoxin, ButaIT （Buthus tamulus insecticidal toxin; red scorpion toxin） linked to the C-terminal region of ASAII or GNA were produced as recombinant proteins （GNA/ ButaIT and ASA/ButaIT） by expression in Pichia pastoris. In both cases the C-terminal sequence of the lectin was truncated to avoid post-translational proteolysis. The GNA- containing fusion protein was toxic by injection to cabbage moth larvae （LD50≈ 250μg/g）, and when fed had a negative effect on survival and growth. It also decreased the survival of cereal aphids （Sitobion avenae; Homoptera） from neonate to adult by 〉70% when fed. In contrast, the ASA-ButaIT fusion protein was non-toxic to aphids, and had no effect on lepidopteran larvae, either when injected or when fed. However, intact ASA-ButaIT fusion protein was present in the haemolymph of cabbage moth larvae following ingestion, showing that transport of the fusion had occurred. The stabilities of GNA/ButaIT and ASA/ButaIT to proteolysis in vivo after injection or ingestion differed, and this may be a factor in determining insecticidal activities.