Production of subtilisin proteases in bacteria and yeast.

In this review, we discuss the progress in the study and modification of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classification, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specifics of) their modification, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i.e., composition of the media, culture duration, and the influence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i.e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modified by genetic engineering and having better properties: e.g., thermal stability, general resistance to detergents and specific resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

Diversity and occurrence of methylotrophic yeasts used in genetic engineering.

Methylotrophic yeasts have been used as the platform for expression of heterologous proteins since the 1980's. They are highly productive and allow producing eukaryotic proteins with an acceptable glycosylation level. The first Pichia pastoris-based system for expression of recombinant protein was developed on the basis of the treeexudate- derived strain obtained in the US southwest. Being distributed free of charge for scientific purposes, this system has become popular around the world. As methylotrophic yeasts were classified in accordance with biomolecular markers, strains used for production of recombinant protein were reclassified as Komagataella phaffii. Although patent legislation suggests free access to these yeasts, they have been distributed on a contract basis. Whereas their status for commercial use is undetermined, the search for alternative stains for expression of recombinant protein continues. Strains of other species of methylotrophic yeasts have been adapted, among which the genus Ogataea representatives prevail. Despite the phylogenetic gap between the genus Ogataea and the genus Komagataella representatives, it turned out possible to use classic vectors and promoters for expression of recombinant protein in all cases. There exist expression systems based on other strains of the genus Komagataella as well as the genus Candida. The potential of these microorganisms for genetic engineering is far from exhausted. Both improvement of existing expression systems and development of new ones on the basis of strains obtained from nature are advantageous. Historically, strains obtained on the southwest of the USA were used as expression systems up to 2009. Currently, expression systems based on strains obtained in Thailand are gaining popularity. Since this group of microorganisms is widely represented around the world both in nature and in urban environments, it may reasonably be expected that new expression systems for recombinant proteins based on strains obtained in other regions of the globe will appear.

[NESPRINS--nuclear envelope proteins ensuring integrity].

This review describes the nesprins (nuclear envelope spectrin-repeat proteins), which are recently discovered family of nuclear envelope proteins. These proteins play an important role in maintaining the cellular architecture and establish the link between the nucleus and other sub-cellular compartments. Many tissue-specific diseases including lipodystrophies, hearing loss, cardiac and skeletal myopathies are associated with nesprins mutations. These proteins comprise of multiple tissue specific isoforms which contain spectrin repeats providing interaction of nesprins with other nuclear membrane proteins, cytoskeleton and intranuclear matrix. We summarize recent findings and suggestions about nesprins structural organization and function inside the cell. Human diseases caused by abnormal nesprins expression are also described.