Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris
2015
Autori:
Tadić, VojinBalaž, Ana Marija J.
Petrić, Marija
Milošević, Snežana
Zelenović, Nevena D.
Raspor, Martin
Tadić, Jovan M.
Prodanović, Radivoje M.
Tip dokumenta:
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt:
We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca
sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia
pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp)
from L. sativa cloned into the vector pUC57 and inserted into plasmids
pYES2 and pGAP using Escherichia coli DH5 alpha strain. The P. pastoris
strain X-33 and the S. cerevisiae strain InvSC1 were used for
extracellular expression of CHO. After transformation of P. pastoris
X-33 with CHO-pGAP construct none of the colonies showed CHO activity.
Two samples displayed a band which did not exist in the sample with the
empty vector similar to the molecular weight of CHO. The S. cerevisiae
strain InvSC1 has been also transformed with CHO-pYES constructs. Three
colonies grew on the plate with cells transformed with the construct.
One of the samples showed a band corresponding to about 110 kDa, but no
CHO activity was recorded in this case either. Cloning of the foreign
genes and heterologous expression in yeasts is widely used in
biotechnology, but sometimes can be very dependent on the gene sequence
and strain used. In order to obtain active CHO enzyme the further
studies on purification and refolding of expressed protein are
necessary.
Ključne reči:
Saccharomyces cerevisiae; Pichia pastoris; carbohydrate oxidase; glycosylationIzvor:
Hemijska Industrija, 2015, 69, 6, 689-701
DOI: 10.2298/HEMIND140823003T
ISSN: 2217-7426