Organizing co mmittee

 
 

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Poster presentation 
183 
CLONNING OF RECOMBINANT PLASMID pPICZαA-TP, 
ENCODING THE THYMIDINE PHOSPHORYLASE IN Pichia 
pastoris YEAST 
 
J.M. Abdurakhmanov, S.A. Sasmakov, Sh.Sh. Hasanov, O.N. Ashirov,
F.B. Eshboev, Kh. Dolimov, S. Gaynazarova, Sh.S. Azimova 
S.Yu. Yunusov Institute of the Chemistry of Plant Substances Academy of sciences of the 
Republic of Uzbekistan st. Mirzo-Ulugbek, 77, 100170 Tashkent 
e-mail: [e-mail], [phone] 
Nowadays, viral, bacterial infections, as well as oncological diseases are the most 
important health problems in the world. The use of modified nucleosides to solve these 
problems is hotly debated by the world's leading scientists. Many preparations based on 
modified nucleosides are obtained by methods of multi-stage chemical synthesis, which 
has a number of significant drawbacks. At the same time, the biotechnological method 
for the synthesis of nucleosides using genetically engineered nucleoside phosphorylases 
makes it possible to replace chemical synthesis with enzymatic synthesis. These 
enzymes simultaneously catalyze the hydrolysis and transglycosylation of carbohydrate-
containing heterocyclic compounds. For this purpose, bacterial cell lysates or purified 
recombinant enzymes are used such as uridine-UP, thymidine-TP, pyrimidine (PyNP), 
purine nucleoside phosphorylase (PNP) or N-deoxyribosyltransferase (N-DRT). Recent 
research results have shown that 
Escherichia coli
thymidine phosphorylase (
Ec
TP, EC 
2.4.2.4) can be successfully used for the biocatalytic derivatization of heterocyclic 
nitrogen containing bases. 
Particularly promising is the production of the thymidine phosphorylase enzyme 
using the 
Pichia pastoris
yeast expression system, which is free of endogenous and 
pyrogenic compounds and has a high ability to synthesize recombinant protein. Based 
on this, the aim of this work is to clone a recombinant plasmid encoding 
Escherichia 
coli
thymidine phosphorylase (
Ec
TP) in the 
Pichia pastoris
expression system. 
During our research, cDNA - thymidine phosphorylase (deoA gene, insert), 1326 bp 
in size, was amplified by PCR using genomic DNA isolated from 
Escherichia coli
strain cells RKMUz – 221 as a template using the following primers (a patent 
application has been filed for these primers): 
1) Forward – 5’- ХХХХХХХTTCATGTTGTTTCTCGCACAA -3’ 
2) Reverse – 5’- ХХХХХХХAGATTATTCGCTGATACGG -3’ 
In the process, FastDigest 
EcoRI
and FastDigest 
XbaI
(Thermo Scientific, USA) 
enzymes were used as restriction enzymes and eventually, based on the transfer vector 
pPICZαA the recombinant plasmid DNA pPICZαA-TP (4862 bp) containing cDNA 
(deoA gene, 1326 bp) of the thymidine phosphorylase (
Ec
TP) of 
E. coli
were 
constructed. 
So, the cloned new plasmid pPICZαA-TP can be used for expression of recombinant 
thymidine phosphorylase (
Ec
TP, EC 2.4.2.4) enzyme in 
Pichia pastoris
yeast. 
Fund: The study was carried out within the framework of project F-FA-2021-360, 
Ministry of Innovative Development of the Republic of Uzbekistan. 

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Poster presentation 
184