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

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...

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Bibliographic Details
Published in:Molecular biotechnology 2021-04, Vol.63 (4), p.316-326
Main Authors: Liew, Yvonne Jing Mei, Lee, Yean Kee, Khalid, Norzulaani, Rahman, Noorsaadah Abd, Tan, Boon Chin
Format: Article
Language:English
Subjects:
Allosteric properties
Binding sites
Bioactive compounds
Biochemistry
Biological Techniques
Biosynthesis
Biotechnology
Cell Biology
Chemical synthesis
Chemistry
Chemistry and Materials Science
E coli
Flavonoids
Holes
Human Genetics
Kinases
Membrane proteins
Membranes
Molecular docking
Natural products
Original Paper
Prenyltransferases
Production methods
Protein biosynthesis
Protein Science
Protein synthesis
Proteins
Systems analysis
Tobacco
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Summary: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.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-021-00304-z