A New Approach to Artificial and Modified Proteins: Theory-based Design, Synthesis in a Cell-free System and Fast Testing of Structural Properties by Radiolabels

A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a ‘halfalbebetin’ (one of these repeats) has also been synthesized and studied. It was shown that ‘half-albebetin’ is also compac

The \u3cem\u3ede Novo\u3c/em\u3e Protein with Grafted Biological Function: Transferring of Interferon Blast-transforming Activity to Albebetin

The de novo protein albebetin has been designed recently to form a predetermined tertiary fold that has not yet been observed in natural proteins. An eight amino acid fragment (131–138) of human interferon α2 carrying the blasttransforming activity of the protein was attached to the N-terminus of albebetin next to its initiatory methionine residue. The gene of chimeric protein was expressed in a wheat germ cell-free translation system and synthesized protein was tested for its compactness and stability. Its ability for receptor binding was also studied. We have shown that albebetin with attached octapeptide is practically as compact as natural proteins of corresponding molecular weight and possesses high stability toward the urea-induced unfolding. It binds murine thymocyte receptor at a high affinity and activates the thymocyte blast transformation efficiently at a concentration of 10-11 M

Virtual In Silico PCR in Two-Dimensional Format as a Tool for Elucidating Phylogenetic Relationship in Allopolyploid Forms with Wheats and Their Wild Relatives Aegilops Used as an Example

Определение природных доноров трех субгеномов BAD мягкой пшеницы (Triticum aestivum L.) имеет большое значение в целях разработки технологий по дальнейшему усовершенствованию данной культуры. До сегодняшнего дня остаются открытыми некоторые вопросы происхождения субгеномов B и A мягкой пшеницы, тогда как донором субгенома D считается Aegilops tauschii подвид strangulata. Для установления видов- доноров субгеномов полиплоидных форм активно применяется сравнение нуклеотидных последовательностей различных генов, а также фрагментов повторяющейся ДНК, что позволяет строить филогенетические древа. Однако в этом случае в анализ берется лишь одна или несколько генетических систем или локусов. Предложенное нами геномное штрихкодирование, не привязанное к какой-либо генетической системе, имеет преимущество, поскольку in silico RAPD-анализ «находит» одинаковые по размеру участки сразу всего генома, делая как бы его полный «срез». Для оценки филогенетического родства разных видов пшеницы использовали метод виртуального мультиплексного RAPD-анализа с 20 ундекамерными праймерами, что позволило создать геномные штрихкоды этих видов, сопровождаемые двумерными картами, составленными для отдельных хромосом анализируемой пшеницы. Предложенный метод компьютерного анализа геномов показал, что T. aestivum субгеном D и Ae. tauschii весьма схожи между собой, что подтверждает их общее происхождение. В случае с донорами субгеномов А и В такие однозначные выводы сделать не удалось, что, возможно, связано с более древним объединением этих субгеномов в тетраплоидной пшенице и накоплением за это время большего количества мутацийIdentification of natural donors of three bread wheat (Triticum aestivum L.) BAD subgenomes is of great importance for developing technologies aimed at improving this crop. The question about the species and subspecies of the wheat–Aegilops alliance serving as donors of the B and A subgenomes remains unanswered, while the Aegilops tauschii subspecies of strangulata is considered as a donor of the D subgenome. To identify the donor species of subgenomes of polypoid forms, the comparison of the nucleotide sequences of various genes, as well as fragments of repetitive DNA, is performed to construct phylogenetic trees. In that case, however, only one or a few genetic systems or loci are taken into analysis. The genomic barcoding proposed by the authors, which is not tied to any genetic system, has an advantage, since in silico RAPD analysis «finds» fragments matching in size in the entire genome, making, as it were, a complete «slice» of it. To estimate the phylogenetic relationship of different wheat species, we used the method of virtual multiplex RAPD analysis with 20 undecamer primers, which made it possible to create genomic barcodes of these species and compile two- dimensional maps for individual chromosomes of the analyzed wheat species. The proposed method of computer analysis of genomes showed that T. aestivum subgenome D and Ae. tauschii are very similar to each other, which may indicate their common origin. No such definite conclusions could be drawn about the donors of subgenomes A and B, which is probably due to the more ancient association of these subgenomes in tetraploid wheat and the accumulation of a larger number of mutations over this tim