[Strategies for Optimizing Recombinant Protein Synthesis in Plant Cells: Classical Approaches and New Directions].

At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.

Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

[Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: a review of basic and applied aspects].

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls sthe development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.).

Two Fix- mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix- (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix- in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix- line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).

[Structural characteristics of lysine-rich histone from the sperm of a mollusk Anodonta piscinalis]. / Osobennosti stroeniia bogatogo lizinom gistona spermy bezzubki Anodonta piscinalis.

Sperm of freshwater bivalve mollusk Anodonta piscinalis was found to contain two fractions of lysine-rich histone: somatic histone H1 and sperm-specific protamine-like histone, named Hp. A detailed analysis of H1 and Hp structure was carried out by means of N-bromosuccinimide, chymotrypsin and pepsin cleavage followed by determination of the lysine residue number, positive charge and molecular length of obtained fragments by the method of incomplete succinylation. It has been shown, that Anodonta histone H1, like the avian histone H5, contains 3 tyrosine residues in the central hydrophobic domain of the molecule. Histone Hp contains 5 tyrosine residues, 3 of which are localized in the hydrophobic domain, while the rest two--in the COOH-terminal part of the molecule, characterized by a strong positive charge. Such unusual disposition of tyrosine residues in the lysine-rich histone has been found for the first time. All the regions of histone Hp molecule contain a great number of arginine residues. The only phenylalanine residue is localised approximately in the middle of the polypeptide chain for both H1 and Hp molecules. On the basis of structure homology between histones H1 and Hp the origin of Hp from H1 in the course of evolution is proposed.

[Determination of lysine residue number, positive charge and molecular lengths of histone H1 and H5 by a method of incomplete succinylation]. / Opredelenie chisla ostatkov lizina, polozhitel'nogo zariada i molekuliarnoi dliny gistonov H1 in H5 metodom nepolnogo suktsinilirovaniia.

A simple method for determination of lysine residue number and positive charge of histones H1 and H5 in the acetic acid -- urea system has been developed. The method is based on incomplete succinylation of lysine residues and allows an accurate determination of protein molecular length. The accuracy of the method has been demonstrated by determination of goose and chicken histones H5 and their fragments. The calibrating curve for determination of molecular lengths of histones H1 and H5 has been plotted. Using the incomplete succinylation method, a detailed analysis of Lymantria dispar histone H1 structure has been carried out. The method under discussion can be used for determining lysine residue number, positive charge and molecular length of practically any protein.

[Histone H1 of reptiles, homologous to histone H5 of birds]. / Giston H1 presmykaiushchikhsia, gomologichnyi gistonu H5 ptits.

Lysine-rich histone H1 of animals from three reptilian orders was studied. Electrophoretically pure H1 histone subfractions were cleaved at residues of tyrosine, methionine, aspartic acid and phenylalanine. The fragments obtained were studied by modified method of incomplete succinylation which permitted to determine the number of lysine residues, the positive charge and molecular length of polypeptides. The structural homology between the fastest reptilia H1 subfraction and avian H5 histone has been shown.

[Molecular variants of histone H5 of linnet (Acanthis flammea)]. / Molekuliarnye varianty gistona H5 chechetki (Acanthis flammea).

Five electrophoretic variants of H5 histone were detected in the population of linnet (Acanthis flammea). Using the method of incomplete succinilation the number of lysine residues, electrophoretic positive charge and molecular length were determined for some variants of H5 histone and for their fragments, obtained after treatment with n-bromosuccinimide and chymotrypsin. The difference in structure of H5 variants was found to be connected with the region, confined by the phenylalanine residue and the C-end of the molecule. The minimal difference in molecular length of fragments carrying the variable region was found to be 6 amino acids, two of them are basic. A series of "regular" fragments was detected after mild treatment with trypsin. The number of these fragments increased in parallel with the increase of histone length. In accordance with the scheme proposed, the difference in structure of linnet H5 variants is caused by insertion of the regular region consisting of tandem repeats (from 3 to 7 times) of the elementary hexapeptide.