Organizing co mmittee

CLONNING OF RECOMBINANT PLASMID pPICZαA-PNP, 
ENCODING THE PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) IN 
PICHIA PASTORIS 
 
J.M. Abdurakhmanov, S.A. Sasmakov, Sh.Sh. Khasanov, O.N. Ashirov,
F.B. Eshboev, Kh. Dolimov, T. Sadullaev, A. Yarilkaganova, 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] 
Modified nucleosides are heterocyclic nitrogenous bases of natural or synthetic origin 
containing monosaccharides - cyclic pentoses. Nucleoside analogs can be synthesized by 
chemical or enzymatic methods, or a combination of these methods. The enzymatic 
transglycosylation / (or) hydrolysis reaction is usually carried out at 50°C to inhibit other 
enzymes such as deaminases. At this temperature, some nucleoside phosphorylases retain 
most of their activity. It has been shown in the literature that 
Escherichia coli
purine 
nucleoside phosphorylase (EC 2.4.2.1) is currently successfully used for the biocatalytic 
preparation of N-β-
D
-ribofuranosyl derivatives of heterocyclic nitrogen-containing bases. 
The use of microorganisms producing nucleoside phosphorylases for the synthesis of 
modified nucleosides of biological and pharmaceutical importance has proved to be 
highly effective. At present, one of the most advanced expression systems that allow to 
obtain recombinant proteins on an industrial scale is the yeast system of 
Pichia pastoris
. 
Considering the above information, the aim of this work is to clone a recombinant 
plasmid encoding 
Escherichia coli
purine nucleoside phosphorylase (PNP) in the 
Pichia 
pastoris
expression system. 
Purine nucleoside phosphorylase cDNA (deoD gene, insert), size of 720 bp was 
amplified by PCR using as a template genomic DNA isolated from cells of 
Escherichia 
coli
strain RKMUz - 221 (the strain was obtained from the collection of industrial 
microorganisms of the Institute of Microbiology of the Academy of Sciences of the 
Republic of Uzbekistan) using the following primers (a patent application has been filed 
for these primers):
1) Forward – 5’- XXXXXXXATGCTACCCCACACATTAATGC -3’ 
2) Reverse – 5’- XXXXXXTTACTCTTTATCGCCCAGCAGAAC -3’ 
The amplificon (insert) was purified by precipitation with 0.5 M magnesium chloride 
(in final concentration 0.05 M) and 96% ethanol (in final concentration 70%). 
One μg of the transfer vector pPICZαA and PCR amplificates were sequentially 
treated with FastDigest 
EcoRI
and FastDigest 
XbaI
restrictases (Thermo Scientific, 
USA). Ligation of the pPICZαA vector with the target gene - purine nucleoside 
phosphorylase was carried out in a volume of 10 μl in a molar ratio of 1:3, respectively, 
using recombinant T4 DNA Ligase (Invitrogen). 
In conclusion, the cloned new plasmid pPICZαA-PNP can be used for expression of 
recombinant purine nucleoside phosphorylase (PNP, EC 2.4.2.1) enzyme in 
Pichia pastoris
. 
Fund: The study was carried out within the framework of project F-FA-2021-360, 
Ministry of Innovative Development of the Republic of Uzbekistan.