Production of human vitronectin in Nicotiana benthamiana using the INPACT hyperexpression platform

Abstract

SummaryHuman vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum‐free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol‐inducible AlcR:alcA gene switch. hVN expression was maximal 4–5 days postactivation of the INPACT platform with a dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of 643 ± 78 mg/kg fresh weight. A three‐stage purification protocol was developed using heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant‐made hVN product of >90% purity. Storage conditions for plant‐made hVN were identified that maximized the capacity of the recombinant protein to promote cell adhesion. Critically, plant‐made hVN was shown to be functionally equivalent to commercial, plasma‐derived hVN at promoting one‐half maximal attachment of murine fibroblast cells (BALB‐C/3T3) in serum‐free medium at <0.1 μg/cm2 to tissue culture plasticware. The INPACT platform represents an attractive means of producing large quantities of functional, animal‐free hVN for in vitro applications.

Localization of Tobacco Yellow Dwarf Virus Replication Using the In Plant Activation (INPACT) Expression Platform

“…While not a significant threat to global food and fiber production, its wide host range and simple genome structure has established TYDV as a useful model for geminivirus research, both as a tool to better understand viral pathogenesis and as a molecular vehicle for gene silencing [28] and manufacturing novel products in planta [29][30][31]. In the latter case, we developed a deconstructed virus vector based on the genome of TYDV termed In Plant Activation (or INPACT) as a means of biopharming a commercially important non-therapeutic protein, human vitronectin, and to express an industrial enzyme and a ribonuclease in Nicotiana species.…”