ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 1, pp. 32–40. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © R.S. Esipov, V.N. Stepanenko, I.O. Zvereva, D.A. Makarov, M.A. Kostromina, T.I. Kostromina, T.I. Muravyova, A.I. Miroshnikov, E.V. Grishin, 2018,
published in Bioorganicheskaya Khimiya, 2018, Vol. 44, No. 1, pp. 38–46.
Biotechnological Method for Production of Recombinant Peptide
Analgesic (Purotoxin-1) from Geolycosa sp. Spider Poison
R. S. Esipov
, V. N. Stepanenko, I. O. Zvereva, D. A. Makarov, M. A. Kostromina, T. I. Kostromina,
T. I. Muravyova, A. I. Miroshnikov, and E. V. Grishin
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
Received May 22, 2017; in final form, June 9, 2017
Abstract⎯Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome.
Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the
purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially
highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four
genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1,
pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp.
was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based
on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization
and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological
purity was developed for preclinical trials.
Keywords: production of recombinant proteins, intein, analgesic peptide, toxin
Severe chronic and sometimes incurable diseases
are frequently accompanied by a pain syndrome. The
relief of pain symptoms in humans is an important
problem of modern medicine. Purotoxin-1 (PT1) iso-
lated from the poison of the Central Asian Geolycosa sp.
spider is potentially a highly efficient drug with an
analgesic effect . The PT1 peptide can selectively
inhibit currents mediated by purinergic P2X3 recep-
tors in sensor neurons in rats, as well as under condi-
tions of the expression of human P2X3 receptor genes
in the HEK 293 cells. It was demonstrated that the
activating effect of natural ATP agonist significantly
decreases under the effect of nanomolar peptide con-
centrations; this is apparently associated with stabili-
zation of the desensitized state of receptors.
Preparations based on PT1 can be effective in the
therapy of pains of different etiology. At present, a
large diversity of low-molecular-weight compounds
modifying the activity of P2X3 receptors is known .
PT1 is the first compound of a polypeptide nature,
which can have a selective modulating effect on P2X3
receptors. The peptide is characterized by an
extremely stable structure (it contains a motif of the
so-called “cystine node”) .
There is a method of PT1 isolation from the natural
source ; in addition, due to small peptide sizes, its
chemical synthesis is possible; however, a biotechno-
logical method for the production of recombinant
PT1 with expression in bacterial cells is the most effi-
cient. Thus, the recombinant PT1 was obtained in the
E. coli BL 21 (DE3) strain with yield of 3 mg from 1 L
of culture . For this, the gene encoding PT1 was
cloned in the composition of the expression pET-32b
vector (Novagen, United States). The desired peptide
was synthesized in the composition of a hybrid pro-
tein, in which thioredoxin was a carrier. The PT1 pep-
tide was cleaved from thioredoxin by means of hydro-
lysis of the catalytic subunit of human enterokinase,
previously produced in the Institute of Bioorganic
Chemistry (Russian Academy of Sciences) . This
method of PT1 production is easily implemented and
justified in a research laboratory; however, it was too
expensive and inconvenient for scaling, since it
included affine chromatography and provided a low
output of the target protein.
In this study, we decided to optimize the above-
described method  and replaced the recognition site
for enterokinase by the recognition site for tobacco
engraving virus (TEV) proteinase, suggesting more
Corresponding author: phone: +7 (495) 366-68-33; e-mail:
Abbreviations: ATP, adenosine triphosphate; CBD, chitin-
binding domain; GyrA, gyrase A; EDTA, ethylenedi-
aminetetraacetic acid; IPTG, isopropyl-β-D-1-thiogalactopy-
ranoside; MES, 2-(N-morpholino)ethanesulfonic acid; PMSF,
phenylmethylsulfonyl f luoride; PT1, purotoxin-1; TRX, thiore-
doxin; ppm, part per million; rpHPLC, reversed-phase high-
performance liquid chromatography; EU, endotoxin unit.