Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda

Liew, Yvonne Jing Mei and Lee, Yean Kee and Khalid, Norzulaani and Abd Rahman, Noorsaadah and Tan, Boon Chin (2021) Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda. Molecular Biotechnology, 63 (4). pp. 316-326. ISSN 1073-6085, DOI https://doi.org/10.1007/s12033-021-00304-z.

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Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.

Item Type: Article
Funders: Fundamental Research Grant Scheme (FRGS/1/2018/STG03/UM/01/2) (FRGS/1/2018/STG03/UM/02/2) (FRGS/1/2016/STG03/UM/01/2), Malaysia Toray Science Foundation (PV022-2016), MyBrain15, Ministry of Education, Malaysia, Postgraduate Research (PPP) Grant (PG073-2016A) (PV005-2017)
Uncontrolled Keywords: Prenyltransferase; In vitro synthesis; Pinostrobin; Liposome; Membrane proteins
Subjects: Q Science > QH Natural history
Q Science > QH Natural history > QH301 Biology
Q Science > QR Microbiology
Depositing User: Ms Zaharah Ramly
Date Deposited: 08 Mar 2022 01:55
Last Modified: 08 Mar 2022 01:55
URI: http://eprints.um.edu.my/id/eprint/27844

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