Zbornik - Društvo genetičara Srbije

76 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Pos-5
Subotica, 30. novembar - 4. decembar 2004.
INAKTIVACIJA GENA ZA PROTEINAZU A U rPAC
PROIZVODNOM SOJU LN5.5 P. pastoris
Lidija Šeneroviæ, Nada Stankoviæ i G. Ljubijankiæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
Pored velike kolièine aktivne PAC u medijumu utvrðeno je Western blot analizom i
prisustvo degradacionih produkata ovog proteina, nastalih delovanjem vakuolarnih
proteaza proizvedenih od strane æelije domaæina. Osim smanjenja ukupne kolièine
intaktnog enzima, usled proteolize oteano je i preèišæavanja rekombinantnog produkta.
Rešenje problema nespecifiène degradacije predstavlja zato znaèajan korak u daljoj
optimizaciji proizvodnje rPAC. Kvašèeva proteinaza A (PrA), vakuolarna aspartièna
proteaza, kodirana je PEP4 genom i sintetiše se u obliku propeptida. Sposobna je za
samoaktivaciju i odgovorna za sazrevanje bar pet vakuolarnih proteaza - proteinaze B
(PrB), karboksipeptidaze Y (CPY), RNKaze, velike aminopeptidaze i nespecifiène
alkalne fosfataze. Zbog toga inaktivacija PEP4 gena ima plejotropni efekat: mutantne
æelije nemaju PrA, CPY i aktivnost alkalne fosfataze, dok je PrB delimièno inaktivirana.
U cilju inaktivacije PEP4 gena P. pastoris, konstruisan je disrupcioni integrativni vektor
pNat PEP4. pNat PEP4 nosi nat1 dominantni marker koji obezbeðuje rezistenciju na
antibiotik nurseotricin i središnji fragment PEP4 gena velièine 400bp, za koji se
oèekivalo da æe putem homologne rekombinacije omoguæiti ugradnju inaktivacione
kasete u PEP4 lokus. Zbog nepostojanja metode kojom bi se direktno merila PrA
aktivnost u æelijama, uraðen je esej koji omoguæuje indirektno utvrðivanje inaktivacije
PEP4 gena preko praæenja aktivnosti CPY (APNE overlay assay). Nekoliko kolonija
koje su pokazale smanjenu aktivnost CPY proverene su dalje Southern blot analizom. Od
8 izabranih kolonija 4 su pokazale uspešnu ugradnju inaktivacione kasete u PEP4 lokus.
INACTIVATION OF A PROTEINASE A GENE
IN P. pastoris LN5.5 STRAIN PRODUCING rPAC
The highest yield of rPAC was obtained in LN5.5 strain with four copies of pac gene integrated
into genom. Appart from the high quantity of secreted active rPAC in medium
considerable amount of degradation products created by the host cell vacuolar proteases
was detected by Western blot. In addition to reducing amount of the intact product in the
medium, proteolysis also complicates the recovery process. Therefore, in further optimization
of the expression of rPAC the issue of nonspecific proteolysis is an important factor.
Proteinase A (PrA), yeast vacuolar aspartyl protease, encoded by PEP4 gene is synthesized
as a propeptide. It is capable of self-activation, as well as subsequent activation
of additional five vacuolar proteases, such as proteinase B (PrB), carboxypeptidase Y
(CPY), RNase, large aminopeptidase and nonspecific alkaline phosphatase. The inactivation
of PEP4 gene results in a pleiotropic phenotype: mutant cells lack PrA, CPY, alkaline
phosphatase activities and partially PrB activity. In order to inactivate PEP4 gene,
integrative vector pNat PEP4 was constructed. pNat PEP4 contains nat1 dominant
marker gene conferring resistance to antibiotic nourseothricin and a 400bp portion of
PEP4 gene for the integration of inactivation cassette by homologous recombination into
host chromosome. Since a satisfactory plate assay that directly measures PrA activity in
colonies is not available, we performed APNE overlay assay which enables detection of
PEP4 inactivation by following CPY activity. Colonies that appeared to have low CPY
activity were selected for Southern blot hybridization analysis. Four of eight selected colonies
showed successful integration of inactivation cassette in PEP4 locus.