Biomedical Engineering Reference
In-Depth Information
Widely used DNA polymerases, such as
Taq
DNA polymerase and its analogs can be
completely inactivated with 0.2% of whole human blood [5, 6]. Since DNA extraction is
costly and time consuming, creation of DNA polymerase resistant to high concentrations of
whole-blood would be very beneficial.
To overcome some of above-mentioned shortcomings of DNA polymerases a special
approach was proposed in literature aimed at production of chimeric proteins incorporating in
addition to polymerase domain a non-specific DNA-binding domain enhancing matrix
affinity of polymerase and, as a result, increasing processivity, synthesis rate, precision and
resistance to high ionic strength of solution [5, 7, 8].
In this study we have engineered
Escherichia coli
strain producing the chimeric protein
which contains Klen
Taq
DNA polymerase (N-terminal 279 amino acids deletion variant of
full length
Taq
DNA polymerase) and additional N-terminal sequence-non-specific DNA
binding domain of
Sulfolobus solfataricus
(SSO7D).
2.
M
ATERIALS AND
M
ETHODS
The fragment of Klen
Taq
DNA polymerase gene (KT) (truncated form of full-length 832
amino acid
Taq
polymerase lacking the N-terminal 279 amino acid portion) was amplified by
PCR from the chromosomal DNA
of
Thermus aquaticus
(ATCC 25104) using the following
synthetic oligonucleotide primers: F (5'-
GAATTC
CTCCTCCACGAGTTCGGCCTTC-3') и R
(5'-TAT
GTCGAC
TTAGTGATGGTGATGGTGATGCTCCTTGGCGGAGAGCCAGT-3').
Eco
RI (F) and
Sal
I (R) restriction sites were inserted at 5′ ends of the primers (the bases are in
italic). The R oligonucleotide contains an additional sequence (shown in bold) encoding a 6-
His affinity tag at the C-terminus of resulting protein to facilitate its purification.
Amplification was performed using
Taq
or
Pfu
DNA polymerase and a PTC-200
GeneAmp PCR system from Bio-Rad (USA). The resulting products were isolated with
Wizard SV gel and PCR clean-up system (Promega, USA), digested with
Eco
RI and
Sal
I
restriction endonucleases, and ligated into the pET22b+ (Novagen, USA) expression vector,
which had been restricted with the same enzymes. The resulting recombinant plasmid was
designated pET22KT.
The nucleotide sequence encoding protein SSO7D (gene
sso7d
) was obtained from NCBI
GenBank [9] (gene ID: 1453539, downloaded in October 2009). Codons for protein
expression in
E. coli
cells
were designed using
DNA 2.0 Gene Designer (USA). The
restriction site for
Nde
I was linked to the 5' end of the gene
sso7d
. The 3' end was elongated
with nucleotide sequence, encoding linker oligopeptide and restriction sites for
Eco
RI. Design
of oligonucleotides to assemble the gene, coding for protein SSO7D, was carried out using
program TmPrime, version 3.0 (http://prime.ibn.a-star.edu.sg). Assembling of the SSO7D
domain was completed by PCR [10].
The resulting PCR product (gene
sso7d
) was digested with
Nde
I and
Eco
RI restriction
endonucleases and ligated into the pET22KT vector, which had been restricted with the same
enzymes.
The resultant recombinant plasmid (designated pET22KT-SSО7D) was introduced into
E. coli
strain BL21(DE3). The transformed cells were grown at 37°C with orbital shaking at
200 rpm in 250 ml Erlenmeyer flasks containing 50 ml of Luria-Bertani (LB) culture
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