Letno poročilo 2005

Laboratorij za biosintezo in biotransformacijo
Laboratory for Biosynthesis and Biotransformation
reakcij teh encimov, predvsem v primerjalno
analizo produktov (do sedaj sta znana dva
mo`na produkta) pri vrsti raznolikih pogojev
encimske reakcije in vitro. Izolirali smo glavni
produkt vseh treh kinaz in ga analizirali z
nuklearno magnetno resonanco (NMR), kar
je do sedaj prva strukturna analiza tega
produkta. [tudije ostalih fizikalno-kemijskih
lastnosti teh molekul so v teku.
- V sklopu raziskav celi~nega cikla pri kvasovki
S. cerevisiae smo pokazali, da je gen ECM11
pomemben del zapletene mre`e genov,
vpletenih v sporulacijo, ~eprav je po podatkih
iz literature uvr{~en med gene, vpletene v
biosintezo celi~ne stene. Ugotovili smo, da v
kvasovki gen ECM11 vpliva na procese mejoti~ne
replikacije, mejoti~ne rekombinacije in
na segregacijo kromosomov. Z analizo na{ih
rezultatov smo ugotovili, da pri sevih z
okvarjenim genom ECM11 pride do napak
pri mejoti~nem prekri`anju, kar je vzrok za
spremembe pri segregaciji kromosomov med
mejozo I in za spremembe pri konverziji
genov v teh sevih. Pokazali smo, da se koli~ina
proteina Ecm11, ki je med mejozo zelo
verjetno vezan na protein Smt3 (sumoiliran),
mo~no pove~a med procesom sporulacije.
- Kljub velikemu {tevilu novih ekspresijskih
sistemov, kvasovka S. cerevisiae ostaja
pomemben gostitelj za pridobivanje razli~nih
heterolognih proteinov. Promotorske sekvence
genov GAL1, GAL7 in GAL10, ki omogo~ajo
visoko ekspresijo teh genov, sodijo
med najbolj preu~ene in so pomembne za
na~rtovanje in konstrukcijo vektorjev. Z lastno
analitsko metodo za kvantitativno preiskovanje
velikega {tevila razli~nih kvasnih
mutant in vivo, smo ugotovili, da sta za
ekspresijo iz promotorja GAL1, najpomembnej{a
proteina Gal1 in Gal4. Z gensko
manipulacijo smo lokus gena GAL1, ki kodira
encim galaktokinaza, zamenjali z genom za
transkripcijski aktivator Gal4. Na~rtovana
zamenjava genov je v mutiranem sevu
povzro~ila bistveno izbolj{anje specifi~ne
produktivnosti rekombinantnih proteinov, ki
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cyclic nucleotides. This project is a collaboration
with a group of Prof. Dr. Visweswariah
from Indian Institute of Science in Bangalore,
India. We have expressed the recombinant
proteins from M. tuberculosis in the laboratory
strain of E. coli. The proteins were produced
and purified in the quantities sufficient
for the crystallographic and other bio-physical
studies. The purified proteins were crystallized
and the determination and analysis
of the crystal structures is currently in
progress.
- Ecm11 is classified as a protein involved in
yeast cell wall biogenesis and organization,
but we provided evidence that it is involved
in meiosis as well. We found out that mutants
with deleted ECM11 exhibit complex
defects in meiosis: replication, recombination
and chromosome segregation are affected.
Summarising our data, we concluded that
the absence of the functional ECM11 gene
product affects meiotic crossing-over resulting
in abnormal gene conversion and chromosome
segregation in meiosis I. Additional
results showed that the amount of Ecm11
protein in the cell is elevated significantly
during meiosis.
- Yeast S. cerevisiae is an attractive host organism
for production of foreign proteins, despite
of large number of newly designed expression
system. Well-studied GAL1, GAL7
and GAL10 promoters, enabling high expression
of these genes, are important for design
and construction of yeast plasmids. With
own analytical procedure for quantitative
screening of large number of yeast mutant
in vivo, we have demonstrated that Gal1 and
Gal4 are the most important proteins for expression
of heterologous genes driven by galactose-inducible
promoter. Using recombinant
DNA techniques, GAL1 gene, encoding
galactokinase, was replaced by transcriptional
activator GAL4. The described replacement
of GAL genes significantly improved
specific productivity of recombinant proteins.
Therefore, newly designed GAL recombinant