Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

Carla Marusic,Giampaolo Buriani,Selene Baschieri,James Nuttall,Lorenzo Frigerio,Chiara Lico,Eugenio Benvenuto,Raffaele Lombardi

doi:10.1186/1472-6750-7-12

Abstract

BackgroundPlants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco.ResultsWe designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure.ConclusionWe have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

Highlights

Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components
We generated a number of constructs for Nef expression in the plant cytosol or secretory pathway (Fig. 1)
Protoplast homogenates were subjected to immunoprecipitation with anti FLAG antiserum

Summary

Introduction

Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Plants have emerged as a safe and economical alternative to mainstream protein expression systems based on the large-scale culture of microbes or animal cells or on transgenic animals to produce biopharmaceuticals. Diverse, complex macromolecules such as antibodies [1,2] and vaccine components [3] have been successfully expressed in plant cells. Insect and mammalian cell cultures represent complex expression platforms requiring expensive procedures and may be contaminated with toxins, viruses or prions, raising concerns on the safety of the final product. The plant secretory pathway, on the other hand, has been shown to be suitable for the production and accumulation of high amounts of heterologous proteins [6,7]

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Conclusion