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Protein Expression and Purification of Taq Polymerase

Jonathan Alexander - Personal Name;

The development and application of molecular diagnostic technologies has triggered a
revolution in infectious disease diagnosis and surveillance over the years. The ability to deliver a quick,
specific, reliable, cost-effective, and straightforward alternative is one of the advantages of using
molecular diagnostics. Taq polymerase is an example of an enzyme that is required in molecular
diagnostics testing. Taq polymerase is a heat-resistant DNA polymerase derived from the
extremophilic bacteria Thermus aquaticus. The purpose of this research is to confirm the presence of
the ""Taq polymerase"" product generated from Escherichia coli BL21(DE3) pLysS bacteria and purify it.
The experiment began with the growth of Escherichia coli BL21(DE3) pLysS bacteria, which had already
been engineered to incorporate the Taq polymerase DNA fragment and linked to the DNA sequence
that encodes the His-tag within the bacteria plasmid. The bacterium sample was in the form of a
glycerol stock, and the bacterial growth was measured using the OD
600
. The culture was then treated
with 1 mM IPTG to induce the expression of the Taq polymerase protein. The protein produced as Taq
polymerase was validated using SDS-PAGE and Western blot techniques. The method was then carried
on to the purification phase, which involved the use of IMAC with Ni-NTA resin. The His-tag is specially
bound to the resin and can assist in the separation of the Taq polymerase protein from other
impurities. The purity percentage of the purified Taq polymerase protein was then determined using
the ImageJ software. Lastly, the buffer after purification was changed into a storage buffer using the
dialysis process, and the final concentration of the purified Taq polymerase was evaluated using
absorbance at 280 nm. The final outcome showed that the Taq polymerase protein was successfully
produced with a high purity percentage of 89%. However, the final protein quantification analysis after
dialysis revealed only a 0.014 mg total protein concentration. As for the recommendation, it is
recommended to continue the study to the activity evaluation step. Another purification step also can
be performed to increase the purity percentage of the purified protein. Analyze the growth curve of
bacteria, try experiments with different duration and concentration of IPTG induction also can be done
in the future to maximize the protein concentration.


Availability
#
Reference Collection (EP Report) EP BT024
EP23059
Available
Detail Information
Series Title
-
Call Number
-
Publisher
i3L, Jakarta : i3L, Jakarta., 2023
Collation
-
Language
English
ISBN/ISSN
-
Classification
NONE
Content Type
-
Media Type
-
Carrier Type
-
Edition
-
Subject(s)
Taq polymerase
protein expression
protein purification
Ni-NTA column
Specific Detail Info
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Statement of Responsibility
-
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Indonesia International Institute for Life Sciences - Learning Resources Center
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i3L Learning Resources Center (LRC) is vital part of your academic experience at Indonesia International Institute for Life-Sciences. LRC exists to support the teaching, learning and research programs of the Institute through the provision of high quality services and facilities which include access to a range of printed and digital resources primarily in the field of life-sciences and business. 

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