Projects / Programmes
Genska tehnologija za fermentacijsko proizvodnjo biološko aktivnih beljakovin (Slovene)
January 1, 1999
- December 31, 2003
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| P320 |
Natural sciences and mathematics |
Nucleic acids, protein synthesis |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
| T490 |
Technological sciences |
Biotechnology |
Researchers (18)
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
20,942 |
Abstract
The proposed research program is based on the long-term experience in biochemical, bio-molecular and biotechnological research of lower eucariotes, especially filamentous fungi. In the seventies and eighties the research group took part in the development of the submerged production of ergot alkaloids by Claviceps fungi, which was transferred to production in the Pharmaceutical and Chemical Company Lek. In the nineties, the group extended its research area to genetic and protein engineering for biosynthesis of biologically active and pharmacologically useful recombinant proteins. Using human tumour necrosis factor alpha (TNF) as a model protein, they succeeded to design, clone and express in E. coli numerous new analogues with newly introduced properties. Many of them have been pre-clinically tested at the Institute of Oncology in Ljubljana, and one of them showing significantly improved therapeutic properties seems to be an excellent candidate for future clinical testing. Lower eucariotes (yeast, filamentous fungi) exhibit numerous advantages for expression of heterologous recombinant proteins, such as high production, ability of post-translational modifications, GRAS status, secretion into the medium, which results in simpler purification, etc. Therefore, the scope of this research program is development of procedures for biosynthesis of highly purified recombinant proteins in the lower eucariote expression-secretion systems. This includes a whole series of research methodologies: from studies of biochemistry and molecular biology of some basic cell life properties of lower eucariotes, design of gene constructs and their synthesis by PCR, site directed mutagenesis, cloning in E. coli, expression of heterologous gene constructs in yeast and filamentous fungi, research of processing and secretion mechanisms, to research of optimisation and scale-up of fermentation, studies on isolation and purification procedures and testing of biological properties of recombinant products. Some basic research will be done on proteins involved in the cell cycle of S. cerevisiae. Computer modelling and relevant r-DNA technologies will be used for elaboration of gene constructs for TNF, G-CSF, EK, GFP, some enzymes of steroid metabolism and respective analogues, which will be expressed in yeast (e.g. P. pastoris) and filamentous fungi (e.g. A. niger) and used as model molecules for studies of secretion mechanisms. Protein engineering will also be applied for research of optimised purification methodologies using engineered purification handles. "In vitro" biological testing of recombinant proteins will be done on mammalian cell cultures, while "in vivo" biological testing of their pharmacological and therapeutic properties will be performed in collaborative institutions.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Final report
