Research Article: Hansenula Polymorpha TERT: 
A Telomerase Catalytic Subunit Isolated in Recombinant Form with Limited Reverse Transcriptase Activity

Date Published: , 2012

Publisher: A.I. Gordeyev

Author(s): E.M. Smekalova, O.A. Petrova, M.I. Zvereva, O.A. Dontsova.



Telomerase is a ribonucleoprotein, the main function of which is to synthesize
telomeres, i.e. repetitive sequences which are localized at the ends of
eukaryotic chromosomes. Telomerase maintains the stability of the genome in
eukaryotic cells by replicating chromosomal ends. The structural and functional
investigation of the telomerase complex is significantly restricted due to
difficulties connected with the isolation of its main catalytic subunit in
recombinant form. Herein, we describe a method developed for the isolation of
the recombinant telomerase reverse transcriptase from thermotolerant
yeastHansenula polymorpha. A functional test performed for
the isolated protein and the RNA/DNA duplex, simulating the interaction of
telomerase RNA and telomere, reveals that the isolated catalytic subunit of
telomerase possesses limited reverse transcriptase activity.

Partial Text

Telomerase is a ribonucleoprotein, the main function of which is to synthesize
telomeres, i.e. repetitive sequences that are localized at the ends of eukaryotic
chromosomes, unable to replicate in accordance with the classical replication
mechanism. Telomerase exhibits activity in cells capable of infinite division, such
as germinal and stem cells, as well as in the majority of malignant tumors (85%). It
is believed that the inhibition of the telomerase catalytic function halts the
maintenance of the telomere length, thereby eliminating the infinite replication
potential of tumor cells. Altogether, it allows telomerase to be considered as a
universal target for various antitumor drugs [1]. The main components of telomerase are the protein, the telomerase
reverse transcriptase (TERT), and the telomerase RNA, the matrix of which serves as
a template for the synthesis of the telomere sequence [2]. One of the main difficulties in studying telomerase is the
low stability of its catalytic subunit isolated in recombinant form [3]. The absence of data on the structure of
telomerase prevents the docking of known substances with a view to searching for the
potential effectors of this enzyme, as well as the difficulties attached to
isolating the full-length functional telomerase reverse transcriptase, which thus
impede the testing of the interactions between pharmacological agents and the
target. At the time of writing, the TERT isolated from Tribolium
castaneum is the only full-length telomerase reverse transcriptase that
has been isolated [4]. The distinctive feature
of this protein is the absence of the N-terminal domain typical for other telomerase
reverse transcriptases. Data on the structure of the N-terminal domain in the
telomerase catalytic subunit of Tetrahymena thermophile and its
RNA-binding domain have been obtained [2,

Cloning of the hpTERT Gene into Various Expression Systems

The gene of the hpTERT protein was cloned under the control of the T7 promotor into
the following three expression systems, with the purpose of further isolation of the
protein from E. coli cells: 1) pCDF with a 6His tag at the
N-terminus of the hpTERT protein; 2) pET33b+ with a 6His tag at the С-terminus
of the hpTERT protein; and 3) pET30aTEV with 6His- and S tags at the N-terminus of
the hpTERT protein. Such location of tags, i.e. from different
sides of the protein, relates to the ability of the amino acid sequence termini to
fold inside a protein globule; the latter is most probably one of the reasons behind
the decrease in the effectiveness of affinity chromatography. The S tag is a short
sequence (4 kDa) which can be used for the stabilization of proteins in a solution.
The expression of proteins was induced by IPTG; the proteins were purified by
metal-chelate chromatography on Ni-NTA-aragose. The results on the isolation of the
proteins expressed by using various constructions are shown in Fig. 1 . The hpTERT protein is
detected in all the samples eluted with Ni-NTA-agarose. The latter indicates that
the selection of thermotolerant yeast as a source for the production of the
telomerase catalytic subunit was successful. However, in the cases occurring when
the pCDF and pET33b+ vectors are used and a tag is located at the N-terminus or
C-terminus or at both ( Figs. 1A, 1B ), a significant amount of
impurities are detected on the resin, along with a target protein. It should be
noted that the amount of impurities relative to the amount of the target protein is
lower when the pET33b+ with a 6His tag at the C-terminus of the protein is used; in
all likelihood, this reflects the close orientation of the N-terminus in hpTERT. The
isolation of the protein with the help of the S tag (the pET30aTEV construction)
gives a much better result ( Fig.
1C ). Apparently, a well-structured short N-terminal S tag
significantly enhances the stability of the protein soluble form. The protein was
isolated and additionally purified by ion-exchange chromatography on SP-sepharose (
Fig. 2 ). The final
characteristics of the protein sample obtained are the following: the concentration
is 5 mg/ml, the yield is 5 mg/L of the E. coli cell culture, and
the content of impurities is not more than 1%.