An Application of pET SUMO Protein Expression System in Escherichia coli : Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein
Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:39 |
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| Enthalten in: |
The protein journal - 39(2020), 1 vom: 16. Feb., Seite 54-61 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Tan, Michelle Siying [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 22.09.2020 Date Revised 10.01.2021 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/s10930-019-09872-1 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM30227362X |
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| 100 | 1 | |a Tan, Michelle Siying |e verfasserin |4 aut | |
| 245 | 1 | 3 | |a An Application of pET SUMO Protein Expression System in Escherichia coli |b Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein |
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| 500 | |a Date Completed 22.09.2020 | ||
| 500 | |a Date Revised 10.01.2021 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Cloning | |
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| 650 | 4 | |a Kras4BG12V | |
| 650 | 4 | |a Protein expression | |
| 650 | 4 | |a Protein purification | |
| 650 | 4 | |a pET SUMO | |
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| 700 | 1 | |a Teh, Yuan Han |e verfasserin |4 aut | |
| 700 | 1 | |a Ho, Kok Lian |e verfasserin |4 aut | |
| 700 | 1 | |a Stanslas, Johnson |e verfasserin |4 aut | |
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