Abstract
Wheat-germ cell-free protein synthesis (WG-CFPS) is a potent platform for the high-yield production of proteins. It is especially of interest for difficult-to-express eukaryotic proteins, such as toxic and transmembrane proteins, and presents an important tool in high-throughput protein screening. Until recently, an assumed drawback of WG-CFPS was a reduced capacity for post-translational modifications. Meanwhile, phosphorylation has been observed in WG-CFPS; yet, authenticity of the respective phosphorylation sites remained unclear. Here we show that a viral membrane protein, the duck hepatitis B virus (DHBV) large envelope protein (DHBs L), produced by WG-CFPS, is phosphorylated upon translation at the same sites as DHBs L produced during DHBV infection of primary hepatocytes. Furthermore, we show that alternative translation initiation of the L protein, previously identified in virus-producing hepatic cells, occurs on WG-CFPS as well. Together, these findings further strengthen the high potential of WG-CFPS to include the reproduction of specific modifications proteins experience in vivo.
Highlights
Wheat germ cell-free protein synthesis (WG-CFPS) is an alternative method to cell-based protein expression
We have recently reported WG-CFPS of DHBs S, which autoassembles during expression in structures called subviral particles (SVPs) (David et al, 2018)
This suggests that the lower band results from alternative translation initiation, which has previously been described to result in two additional DHBs L bands, p33 and p30 (Fernholz et al, 1993)
Summary
Wheat germ cell-free protein synthesis (WG-CFPS) is an alternative method to cell-based protein expression. Reduced or even lacking posttranslational modifications have long been considered a drawback of WG-CFPS compared to other systems, as small cofactors required for the activity of certain enzymes might be eliminated during extract preparation. Most kinases do not need cofactors, and a proteomics study on wheat germs from Triticum aestivum has highlighted the presence of at least 12 different kinases which do not require cofactors for activity, including several serine/threonine kinases (Mak et al, 2006). Even though the exact enzyme content of cell-free extracts from wheat germ remains to be determined, altogether these data suggest that phosphorylation can in principle occur
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