Ectopic protein bodies , a novel strategy for oral delivery of recombinant proteins

SUMMARY Protein bodies are naturally occurring seed storage organelles. The protein sequences responsible for the assembly of such bodies have been used as fusion partner to boost the overall yield of several recombinant pharmaceutical proteins. We have mutated these sequences to add cell penetrating properties in order to make them even more attractive as oral delivery systems. INTRODUCTION Seed storage proteins are produced and stored in a very stable and efficient way within protein bodies (PBs). Such storage organelles constitute a very attractive model for the generation of new drug delivery systems. Naturally occurring storage proteins have been used as fusion partner to induce the packing of the resulting chimeric proteins in storage bodies (1). One well-known fusion protein is gamma zein, which is the major storage protein in maize. The first 90 amino acids are crucial for PB formation and comprise 3 domains: an N-terminal domain comprising two essential cysteins, a repeat domain bearing 8 repeats of PPPVHL and a pX domain harbouring 4 cysteins. Interchain cross linking by disulphide bond formation between the two cysteins (Nand C-terminal) as well as the amphipathicity of the PPPVHL domain that facilitates lateral protein-protein interaction have been identified as crucial factors in the formation of large polymers, namely protein bodies (2). PBs are promising candidates for the delivery of oral pharmaceuticals. They offer a stable environment and are only slowly digested, resulting in a constant uptake of the pharmaceutical over the whole gastrointestinal tract (3). The high protein stability can be combined with improved cellular uptake by using so-called cell penetrating peptides. Aiming to generate PBs, with both extreme stability and increased cell permeability, different mutations were introduced in the gamma zein sequence. EXPERIMENTAL METHODS Two different constructs were designed. In construct Z3, the histidines of the repeat domain were replaced with arginines. The second construct (P1) was designed to keep the hydration potential constant even when histidines were exchanged with arginines. Therefore, 6 repeats (PPPVRL) were used instead of 8 repeats (PPPVHL). Moreover the pX domain was knocked out and replaced with 4 amino acids (CPPC). Both constructs were fused to the fluorescent protein DsRed and then used for agroco-infiltration together with a silencing inhibitor (HcPRO). After 7 days, leaf samples were fixed and processed for immunolocalization. The remaining leaves were harvested and analyzed for their protein content via westernblot. PBs were isolated with extraction buffer (10mM Tris-HCl, 0,4M sucrose, pH 7.5) (1:1 v/v) and purified via a sucrose gradient (3M-1,5M). An in vitro digestion study was performed in order to test the stability of the protein bodies against pepsin and trypsin for various time periods at 37 °C as described by Lee and Hamaker (4). Moreover the purified protein bodies were incubated with CHO cells and analyzed by flow cytometry . RESULTS AND DISCUSSION The mutated versions of gamma zein still possess the ability to form protein bodies, as shown by confocal microscopy of transfected protoplasts and infiltrated leaves (figure 1).