Laboratory of Biotechnology and Molecular Bioengineering

Scientific Head: Grinchenko Andrey Viktorovich, senior researcher, Cand. Sci. Biol., has managed the Laboratory since its establishment in May 2024.

The Laboratory has 12 staff members.

Main fields of research

• Search for and study of the structural and functional properties of biopolymers from marine organisms.
• Biotechnology of marine producers of valuable biologically active compounds.
• Rational design and bioengineering of proteins.
• Development of technologies and tools for medical and aquatic veterinary use.

Approaches and methods used

Rational design in protein bioengineering for personalized medicine requires an in-depth study of protein structural and functional properties and, therefore, an integrated approach that utilizes knowledge and skills in multiple related fields of biology, biophysics, and fundamental medicine. Search for natural protein candidates can currently be conducted using both biochemical methods and bioinformatics approaches. The production of recombinant forms and their targeted mutagenesis requires skills in molecular biology and biotechnology, particularly genetic engineering. Modern analysis of protein functional properties, in addition to biochemistry and molecular biology, requires employment of cellular technologies, especially for the development of medical devices for various purposes. Furthermore, the development of diagnostic systems is directly linked to understanding of pathogenesis. The search for and production of carbohydrate-binding proteins capable of recognizing aberrant glycosylation in tumor cells is impossible without modern knowledge and skills in cell biology, medical biochemistry, and glycobiology. Structural analysis of biopolymers, primarily related to biophysics, is crucial in the rational design of recombinant proteins as a tool for testing and predicting potential changes in their properties. Among the approaches and methods used in the Laboratory are the following:

1. Screening methods for determining the agglutinating activity and carbohydrate specificity of carbohydrate-binding proteins (hemagglutination assay (HA), hemagglutination inhibition assay (HIA), frontal affinity chromatography, and lectin microarrays).
2. Chromatographic approaches to isolation and purification of carbohydrate-binding proteins (affinity chromatography, ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography).
3. Routine methods for determining the polypeptide composition, amino acid sequences, and oligomeric states of carbohydrate-binding proteins (denaturing/non-denaturing polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry).
4. Biophysical methods for quantification of the carbohydrate specificity of carbohydrate-binding proteins toward carbohydrate ligands (surface plasmon resonance, high-resolution single-molecule atomic force microscopy, IR spectroscopy, and thermal shift analysis of protein–ligand complexes).
5. Methods for determining the spatial structure of carbohydrate-binding proteins and their complexes with carbohydrate ligands (high-resolution single-crystal X-ray diffraction, small-angle X-ray scattering, and nuclear magnetic resonance spectroscopy).
6. Methods for identifying aberrant glycosylation patterns in tumor cells (bioinformatics-based searches for glycan signatures using transcriptomic and glycoproteomic data, lectin affinity chromatography, ultra-high-performance chromatography coupled with high-resolution mass spectrometry, and immunoblotting).
7. Protein engineering methods (production of recombinant forms of carbohydrate-binding proteins in various expression systems, development of libraries of mutant carbohydrate-binding proteins using rational design, random, and site-directed mutagenesis).
8. Chemical modification methods for carbohydrate-binding proteins (conjugation with fluorescent carriers).
9. Microscopy methods (light, fluorescence, laser scanning, and electron microscopy).

Many marine organisms are already used as sources of biotechnologically valuable biopolymers, including proteins and polysaccharides. Carbohydrate-binding proteins are found in all organisms, from viruses, bacteria, and protozoans to angiosperms and higher vertebrates. Given the rapid advancement of biomedical research, carbohydrate-binding agglutinin proteins from invertebrates are of particular interest. Previously, these proteins were classified only as lectins; advances in genomic research have significantly extended the list of lectin-like protein groups. Representatives of the phylum Mollusca, in particular bivalves, are especially noteworthy in this regard, as they exhibit an extremely high diversity of structures encoding carbohydrate-binding proteins. The researchers of the Laboratory have already screened the carbohydrate specificity of agglutinins from representatives of such taxonomic groups as Polychaeta, Sipuncula, Thecostraca, Malacostraca, Holothuroidea, and Asteroidea and are planning to extend this list.

Geography of research

The research focus is primarily on the marine waters of the Russian Far East, particularly the coastal waters of the Sea of ​​Japan, which are characterized by exceptional biodiversity and the uniqueness of some of the species found there. However, the structural and functional properties of biopolymers from marine organisms from other regions, including deep-sea species, can also be explored.

The Laboratory researchers have access to the shared-use equipment of the NSCMB FEB RAS, to the instruments of the Far Eastern Center for Electron Microscopy Shared Equipment Facility, the Primorsky Aquarium Shared Equipment Facility, and to the equipped laboratory facilities of the Vostok Marine Biological Station.

The laboratory was established in May 2024 as part of the federal project “Development of Human Capital in the Interests of Regions, Industries, and the Research and Development Sector” within the Science and Universities national project based on the selection of 2023 applications for creation of youth laboratories.

Current research

The biological diversity of the world’s oceans is the source of a vast variety of chemical compounds whose structures and various functional properties have formed over the long course of evolution in the marine environment. Among the many low-molecular-weight compounds isolated from marine biota, a significant number have been discovered and have found practical application in medicine, pharmaceuticals, and biotechnology, including some molecules whose fundamentally novel organization has enabled the development of cutting-edge agents for anticancer therapy and regenerative medicine. High-molecular-weight compounds from marine organisms occupy a special place, among which carbohydrates of various structures and proteins are of particular importance. Together, these proteins and carbohydrates provide molecular recognition mechanisms involved in intercellular association, signaling, and interactions with the extracellular matrix and environmental components.

The molecular recognition mechanisms, in which specialized proteins specifically bind carbohydrates, glycans, and glycoconjugates, enabled the development of multicellularity, the integration of cells with each other, and the formation of fundamental mechanisms of immune defense. In the absence of immunoglobulins, carbohydrate-binding proteins, originally referred to as lectins but now comprising several independent groups of structurally heterogeneous biopolymers, became the basis of immunity in many invertebrates, and the genomes of individual taxonomic groups contain hundreds of genes encoding these proteins. Search for new molecules from this group and study of their structural and functional properties are of great practical interest, as they can be used to create new tools for personalized medicine and veterinary science.

In particular, such molecules could be used to develop diagnostic systems for cancer diseases, as these proteins are capable of recognizing molecular patterns of aberrant glycosylation in cells and tissues, enabling the identification of certain tumor cell phenotypes. Glycobiology, which comprehensively studies the biological role of carbohydrates of various structures, a group of chemical compounds that was previously greatly underestimated, has recently become a separate and actively developing area of research. Many carbohydrates isolated from marine organisms have already found a variety of practical applications, and the study of the structural and functional mechanisms of carbohydrate–protein interactions is of fundamental interest and supports the development of biotechnology and biomedicine.

The main interests of the newly created research team focus on the study of biopolymers from marine organisms, the biotechnology of their producers, and the creation of solutions based on the principles of molecular bioengineering that will contribute to the development of biomedical and veterinary technologies, and also personalized medicine technologies, which are among the priority areas of scientific and technological development in Russia.

For the period 2024–2026, the laboratory’s activities are based on the project “Structural and Functional Properties and Molecular Bioengineering of Carbohydrate-Binding Proteins of Marine Organisms for the Creation of Biomedical Technologies.” The project aims to obtain, rationally design, and characterize the structural and functional properties of marine proteins capable of recognizing specific glycosylation patterns in biostructures. This will facilitate the advancement of personalized medicine technologies and the formation of expertise in synchrotron research on biologically active molecules.

The main objectives are as follows:

search for natural carbohydrate-binding proteins and their coding structures that are promising for biotechnology and biomedicine purposes; production of recombinant forms of candidate proteins; study of structural and functional properties of natural and recombinant carbohydrate-binding proteins; creation of prototypes of molecular recognition systems for biomedical purposes based on original preparations of carbohydrate-binding proteins; formation of a team of researchers capable of combining various methods for bioengineering and rational design of natural biopolymers; and advanced structural and functional synchrotron studies aimed at obtaining biotechnologically valuable products that contribute to the development of personalized medicine.

The Laboratory of Biotechnology and Molecular Bioengineering at the NSCMB FEB RAS maintains close ties with the Laboratory of Biomedical Cell Technologies and the Center for Genomic and Regenerative Medicine (headed by Vadim Vladimirovich Kumeiko, Cand. Sci. Biol.) at the Far Eastern Federal University (FEFU, Vladivostok, Russia). This collaboration involves a detailed characterization of the morphological and cytotoxic properties of the biopolymers being studied. Specifically, a work is underway to evaluate the effects of the resulting proteins on eukaryotic cells using various cell lines from the FEFU collection, including standard cell cultures of various embryoblasts (mesenchymal, epithelial, and neuroepithelial), a panel of cultures with induced driver mutations critical for tumor growth and development, and a unique collection of primary cell cultures obtained from patient tissue samples. The Laboratory researchers also have direct access to the unique equipment at the FEFU.

For example, cell culturing can be performed on the CompacT SelecT robotic autonomous cell culturing system (TAP Biosystems) equipped with modules for determining confluency and viability of cell lines and also on the Cell-IQ continuous image acquisition and analysis system (CM Technologies) which can assess cell proliferation and migration by the machine pattern recognition technology. The influence of biopolymers on model cell systems can be studied using confocal and multiphoton laser microscopy with a FluoView FV1200MPE (Olympus) deep optical imaging system for biomaterials. This system is equipped with an incubator for long-term intravital studies and minimizes phototoxic effects on the cells under study due to highly sensitive detectors and the use of a femtosecond infrared laser. It is also capable of performing total internal reflection fluorescence microscopy (TIRF), which allows real-time intravital acquisition of high-contrast images of structures near the cell membrane in the area immediately adjacent to the glass where total internal reflection occurs. The nanomechanical, rheological, and morphological properties of nanoscale systems, and also protein–cell surface interaction forces can be studied using a Bioscope Resolve atomic force microscope (Bruker), built on a Zeiss Axio Observer A1 inverted fluorescence microscope and equipped with a FluidFM microfluidic system. Protein–ligand interaction affinity and constants can be assessed by both atomic force microscopy and surface plasmon resonance on a Biacore X100 system (General Electric).

Collaboration with the Laboratory for Digital Controlled Drugs and Theranostics at the Federal Research Center “Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences” (headed by Anna Sergeevna Kichkaylo, Dr. Sci. Biol.) will enable the transfer of expertise and adaptation of the experience in the development of diagnostic and therapeutic systems based on the targeted selection of DNA aptamers for biological targets that serve as biomarkers of glioblastoma, lung cancer, and breast cancer cells, as applied to the diagnostic potential of carbohydrate-binding proteins. The expertise of the Laboratory for Digital Controlled Drugs and Theranostics in studying the structure of nucleic acids and their complexes with other biopolymers using small-angle scattering (SAXS) will be applied in the structural analysis of carbohydrate-binding proteins, while their expertise in molecular modeling and molecular docking will facilitate the rational design of these proteins.