Expression of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris


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Abstract

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia рastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).

About the authors

S. S. Terekhov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997

T. V. Bobik

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997

Yu. A. Mokrushina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997

A. V. Stepanova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997

N. M. Aleksandrova

Kazan (Volga Region) Federal University

Email: gabibov@mx.ibch.ru
Russian Federation, Kazan, 420000

I. V. Smirnov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Kazan (Volga Region) Federal University

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997; Kazan, 420000

A. A. Belogurov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Kazan (Volga Region) Federal University

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997; Kazan, 420000

N. A. Ponomarenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997

A. G. Gabibov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Kazan (Volga Region) Federal University

Author for correspondence.
Email: gabibov@mx.ibch.ru
Russian Federation, Moscow, 117997; Kazan, 420000

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