Chemical Modification of Fusion Protein Based on the Thermus thermophilus GroEL Chaperon with AEBSF Protease Inhibitor

Protease inhibitors are routinely used to prepare functional, full-size proteins. Here, we describe modifications of the chimeric protein based on the GroEL chaperon from Thermus thermophilus. Modifications of this chimeric protein resulted from its interaction during sample preparation with an irreversible inhibitor of serine proteases, 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), which belongs to the sulfonyl-fluoride class of compounds. Protein samples were then identified via MALDI-TOF/TOF after in-gel preparation with trypsin. Modifications of tyrosine and lysine amino acid residues were shown to be present. Also, the availability of the tyrosine residue was found to be a prerequisite for its modification. © 2019, Pleiades Publishing, Inc

Химическая модификация слитого белка на основе шаперона GroEL из Thermus thermophilus ингибитором протеаз AEBSF

Production of functional full-size proteins is often involving the use of protease inhibitors. Modifications due to the interaction of the protein with the common, irreversible inhibitor of the sulfonyl fluoride class of 4-(2-amino-ethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) were registered and described during the sample preparation. The obtained forms of the protein after preliminary purification were studied by the MALDI-TOF-TOF method with trypsinolysis from a gel. The dependence of the presence of a modification of the amino acid residue of tyrosine on the degree of its availability for the reaction mixture is shown.Получение функциональных полноразмерных белков чаще всего подразумевает использование ингибиторов протеаз. Зарегистрированы и описаны модификации химерного белка на основе шаперона GroEL Thermus thermophilus, вызванные взаимодействием с необратимым ингибитором сериновых протеаз класса сульфонилфторидов – 4-(2-Аминоэтил)-бензолсульфонилфторид-гидрохлорида (AEBSF) в ходе пробоподготовки. Полученные формы белка после предварительной очистки были изучены методом MALDI-TOF-TOF с трипсинолизом из геля для идентификации. Показано наличие модификаций аминокислотных остатков тирозина и лизина, а также зависимость модификации от степени доступности аминокислотного остатка тирозина

Physicochemical Characteristics of a Variant of Chaperon GroEL Apical Domain Designed to Enhance the Expression and Stability of Target Proteins

Abstract: This work describes the properties of a new protein, a modification of GroEL apical domain designed to be a leader in fusion systems. This polypeptide leader demonstrates a high level of expression in a bacterial system; it is soluble and retains its solubility during standard biochemical manipulations. The secondary structure of the protein and its thermostability, as well as the protein solubility, were studied in a wide temperature range. To simplify the subsequent purification of the target protein, the possibility of its chemical cleavage from the fused protein by methionine residues with cyanogen bromide is provided. © 2019, Pleiades Publishing, Inc

Physicochemical characteristics of modification of chaperon groel apical domain designed to enhance expression and stability of target proteins [Физико-химическая характеристика варианта апикального домена шаперона GroEL для повышения уровня биосинтеза и увеличения стабильности целевых белков]

Описаны свойства нового белка, являющегося вариантом апикального домена шаперона GroEL, предназначенного служить лидером при создании слитых белков. Данный полипептидный лидер демонстрирует высокий уровень биосинтеза в бактериальной системе, растворим и сохраняет растворимость после стандартных биохимических манипуляций. Исследована его вторичная структура и ее термостабильность, а также растворимость белка в широком диапазоне температур. Для упрощения процедуры последующей очистки целевого белка предусмотрена возможность его химического отщепления по остатку метионина под действием бромциана.The expression of heterologous proteins in bacterial system is often impeded or even impossible due, for instance, to their lability, hydrophobicity or toxicity. In many cases, the problem can be partially or completely solved by creating fusion proteins with a leader able to enhance the solubility or stability of a target protein. In this work, the properties of a new protein, a modification of GroEL apical domain, designed to be a leader in fusion systems have been described. This polypeptide leader demonstrates a high level of expression in the bacterial system; it is soluble and retains its solubility after standard biochemical manipulations. The secondary structure of the protein and its thermal stability, and also the protein solubility were studied in the wide temperature range. To simplify the following purification of the target protein, a possibility of its chemical cleavage from the fused protein at the methionine residues using cyan bromide is provided. © 2009-2017 State Research Institute for Genetics and Selection of Industrial