title: Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.
abstract: Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)32tag (32 tandem repeats of "Ser-Pro" motif). The (SP)32tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, 72401, USA.
authors.forename: Ningning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, Arkansas State University, Jonesboro, AR, 72401, USA.
authors.forename: Maureen
authors.initials: M
authors.lastname: Dolan
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, 72401, USA.
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.affiliation: College of Agriculture and Technology, Arkansas State University, Jonesboro, AR, 72401, USA.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Phillips
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, 72401, USA. jxu@astate.edu.|College of Agriculture and Technology, Arkansas State University, Jonesboro, AR, 72401, USA. jxu@astate.edu.
authors.forename: Jianfeng
authors.identifier: http://orcid.org/0000-0002-0106-2516
authors.initials: J
authors.lastname: Xu
mesh_terms: D018533:Biomass; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry*; D004151:Dipeptides / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D009584:Nitrogen / Q000494:pharmacology; D059828:Plant Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology*
chemical_list: D003470:Culture Media; D004151:Dipeptides; D006020:Glycopeptides; D009566:Nitrates; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C465983:seryl-proline; D049452:Green Fluorescent Proteins; D009584:Nitrogen; C006568:ammonium nitrate
keywords: Hydroxyproline-O-glycosylation; Medium manipulation; Nitrogen supply; Plant cell culture; Protein secretion; Recombinant proteins