Characterisation of bacterially expressed structural protein E2 of hepatitis C virus

The E2 glycoprotein is a structural component of the hepatitis C virus (HCV) virion. It interacts with putative cellular receptors, elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti-HCV vaccine. Despite this, relatively little is known about this protein. Previous studies have focused on the antigenic and functional analysis of the glycosylated forms. This report describes expression of the ectodomain of E2 (recE2) in Escherichia coli cells, its purification, and initial characterisation of its structural and functional properties. It is demonstrated that the purified protein forms small soluble aggregates, which retain functional characteristics of its native counterpart, i.e., it interacts with a putative cellular receptor, CD81, and is recognised by both conformation-dependent and -independent anti-E2 monoclonal antibodies

Dimerization of the antimicrobial peptide polyphemusin I into one polypeptide chain: Theoretical and practical consequences

A strategy of sequential dimerization of monomers of antimicrobial peptides (AMPs) into one polypeptide chain has been implemented on the example of a beta-structural AMP polyphemusin I which is one of the most effective candidate for use as an antibiotic. The possible polyphemusin I monomer and dimer structures in lipid membrane were studied in this work via molecular modeling. To this end, these molecules were chemically synthesized so that the dimer represented two monomers connected in series into one polypeptide chain with a flexible linker. The antimicrobial effects of monomer and dimer were then tested on various bacterial cultures, and their similarity was shown. Therefore, we can conclude that the pore formation is not a putative mechanism of the polyphemusin I action. © 2019 State Research Institute for Genetics and Selection of Industrial Microorganisms

Design and Characterization of a Methionineless Variant of Thermostable Chaperon GroEL from Thermus thermophilus

Abstract: The GroEL chaperon is of significant interest due to its structure and function, which evolved to facilitate protein folding, and its potential application in artificial expression systems to obtain soluble recombinant proteins. A GroEL variant derived from Thermus thermophilus in which all methionine residues were substituted for leucine was created. This modified chaperon was purified to homogeneity. GroEL completely retained its quaternary structure of the original chaperon (double heptamer), together with its thermostable properties. Both the structure of the new chaperon and its usage as a carrier to obtain target recombinant proteins are attractive for researchers in the field. Also, the substitution of methionine residues in a carrier protein substantially simplifies target protein purification. © 2019, Pleiades Publishing, Inc

Получение и характеристика безметионинового варианта термостабильного шаперона GroEL из Thermus thermophilus

Chaperone GroEL is the subject of extensive studies concerning its organization and functioning at folding cell proteins as well as the possibility of using it when developing new expression systems to obtain recombinant proteins in soluble forms. In this work, a new variant of Thermus thermophilus chaperone GroEL is developed, in which all methionine residues are substituted for leucine residues. Obtained variant of the chaperone was purified to homogeneity. Modified GroEL retained tetruary structure characteristic for initial chaperone consisting of double heptamer, and also retained the initial thermostability. Obtained variant of the chaperone is interesting not only from the point of view of GroEL structural organization, but it can be effectively used as a leader to obtain recombinant target proteins. The substitution of methionine residues in a leader allows to significantly simplify the following purification of target polypeptide.Шаперон GroEL является объектом интенсивных исследований как с точки зрения его устройства и функционирования при сворачивании белков в клетке, так и возможностей использования в создаваемых системах экспрессии для получения рекомбинантных белков в растворимых формах. Создан вариант шаперона GroEL из Thermus thermophilus, в котором все остатки метионина заменены на остатки лейцина. Полученный вариант шаперона был очищен до гомогенного состояния. Измененный GroEL полностью сохранял четвертичную структуру, присущую исходному шаперону, в виде частицы из двойного гептамера, а также сохранял исходную термостабильность. Полученный вариант шаперона интересен не только с точки зрения структурной организации GroEL, но и может быть эффективно использован в качестве белка-носителя при получении целевых рекомбинантных белков. Замена остатков метионина в белке-носителе позволяет существенно упростить последующие процедуры очистки целевых полипептидов

Dimerization of Antimicrobial Peptide Polyphemusin I into One Polypeptide Chain: Theoretical and Practical Consequences

The study of antimicrobial peptides (AMPs), which are considered a source of new antibiotics, have recently attracted more and more attention. AMPs combine several large groups of peptides with different spectra and different mechanisms of action on bacterial cells. Peptides that form alpha-helices are better studied; the mechanisms of antimicrobial action of peptides that form beta structures have been studied to a lesser extent. A further increase in AMPs bactericidal effect would be useful for the practical use of AMPs. One strategy used to increase the bactericidal effect is the dimerization of AMP, which is usually carried out via the chemical conjugation of monomers. In this work, the strategy of sequential monomer dimerization into a single polypeptide chain was applied with the example of beta-structured AMP polyphemusin I. Polyphemusin I is one of the most effective AMPs and, accordingly, a candidate for practical use as an antibiotic. Several possible mechanisms of its action have been described. The most developed of these are the formation of pores in the cell membrane and translocation through the membrane without pronounced damage. These mechanisms result in different predictions of the effect of polyphemusin I dimerization on its bactericidal properties. In this article, the possible structures of the monomer and dimer of polyphemusin I in the lipid membrane were analyzed with molecular modeling methods. A monomer and a dimer of polyphemusin I, in which two monomers are connected in series through a flexible linker into a single polypeptide chain, were chemically synthesized. Quantitative assessment of the antimicrobial action of the monomer and dimer on different bacterial cultures was carried out; no differences were observed. Combination of the results of molecular modeling and laboratory studies show that pore formation is not a likely mechanism of the action of polyphemusin I. © 2020, Pleiades Publishing, Inc

Physico-Chemical Characterization of Permutated Variants of Chaperone GroEL Apical Domain

Abstract: At biosynthetic production of hydrophobic recombinant proteins there is often a problem of their aggregation in so-called inclusion bodies, after which these proteins are difficult to renature. Obtaining such proteins in soluble forms is a formidable obstacle on the way of using them. One of possible solutions for this problem is the creation of fusion constructs with a leading protein. Previously, we have developed a system for biosynthetic production of insoluble hydrophobic proteins as a part of a fusion construct with a leader based on chaperone GroEL apical domain (GrAD, GroEL Apical Domain). Expressed as a part of a fusion with GrAD, two initially insoluble proteins were successfully obtained in soluble form. Still, such a system may have limitations in use, because supporting hydrophobic proteins in soluble state requires their interaction with GrAD substrate binding surface, which implies correct mutual orientation of proteins constituent parts of the construct, and for some target proteins such interaction may be sterically impeded. To enlarge the capability of using GrAD, the strategy of making permutations was used, which consists in linking original GrAD N- and C-termini with a linker and creating new N- and C-termini situated in closer proximity to the GrAD substrate binding surface. This work describes production and the study of physico-chemical properties of two permutated GrAD variants intended to be used as leaders in fusion constructs. © 2019, Pleiades Publishing, Inc

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