Expression and purification of soluble bacterially-expressed human hexokinase II in E.coli system
Human hexokinase II (HKII) is one of the key enzymes in the glycolytic pathway. It has been postulated that HKII is a potential target for anti-dengue (DENV) drug development, as well as involved in cancer and tumor cell growth. In this work, the human hexokinase II (HKII) gene was cloned into p...
| Main Authors: | , |
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| Format: | Proceeding Paper |
| Language: | English English |
| Published: |
Association for Computing Machinery
2019
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/79728/ http://irep.iium.edu.my/79728/9/79728_Expression%20and%20Purification%20of%20Soluble_new_complete.pdf http://irep.iium.edu.my/79728/2/79728_Expression%20and%20Purification%20of%20Soluble%20Bacterially-Expressed_scopus.pdf |
| Summary: | Human hexokinase II (HKII) is one of the key enzymes in the
glycolytic pathway. It has been postulated that HKII is a potential
target for anti-dengue (DENV) drug development, as well as
involved in cancer and tumor cell growth. In this work, the
human hexokinase II (HKII) gene was cloned into pETite N-His
SUMO vector and transformed into the E.coli strain HI-control
10G for the propagation of clones. Two different expression
hosts, E.coli HI-controlTM BL21 (DE3) and BL21 (DE3) pLysS
were used to optimize HKII expression. In order to obtain the
soluble recombinant HKII in a functional form, we optimized
protein expression at three different temperatures; 17°C, 25°C and
37°C, at 24 hours incubation time. The soluble protein was
expressed in the presence of 0.5 mM isopropyl-2-Dthiogalactopyranoside (IPTG) in TB media at 17°C for 24 hrs.
The expressed protein was then purified to homogeneity by a
combination of Immobilized Metal Ion Affinity Chromatography
(IMAC), size exclusion chromatography (SEC) and ion-exchange
chromatography (IEX), resulting in pure bacterially-expressed
HK2. Taken together, this study has successfully produced
soluble bacterially-expressed human HKII that can be utilized for
further therapeutic studies. |
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