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.
Highlights
Human vitronectin, formerly known as serum spreading factor, is one of the major cell adhesion proteins found in the blood and the extracellular matrix (Conlan et al, 1988; Shaffer et al, 1984)
We recently described the In Plant ACTivation (INPACT) platform, an inducible, high-level expression system for transgenic plants based upon the disaggregated DNA genome of a geminivirus, Tobacco yellow dwarf virus (TYDV) (Dugdale et al, 2013, 2014)
Synchronized activation of transgene amplification and expression from the INPACT platform is strongly dependent on the regulated expression of Rep/RepA activator genes
Summary
Human vitronectin (hVN), formerly known as serum spreading factor, is one of the major cell adhesion proteins found in the blood and the extracellular matrix (Conlan et al, 1988; Shaffer et al, 1984). HVN interacts with diverse proteins via conserved regions located throughout the polypeptide chain (Jin and Varner, 2004; Preissner, 1991; Zhou et al, 2003) and the carboxyl-terminal region contains a cryptic, arginine-rich domain that binds heparin, an anticoagulant. This domain is only exposed after hVN binding to the thrombin–antithrombin III complex in vivo or denaturation with urea, heat, or acid in vitro, and it is this conformational change that activates self-association of hVN into multimers with significantly increased affinity for heparin compared to the monomeric form (Preissner, 1991). The protein must undergo strict regulatory testing for infectious agents and has become prohibitively expensive for applications in translational animal research
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