Development of Pichia pastoris as a rumen escape vehicle for the intestinal delivery of recombinant proteins in ruminants

Abstract

The effectiveness of cellular encapsulation as a method for delivery of bioactive proteins and limiting amino acids to the small intestine of ruminants was investigated. Intracellular expression of green fluorescent protein variant (GFPuv) in Pichia pastoris was used as a visible marker to assess the cellular integrity of P. pastoris and determine the potential of this approach for protecting recombinant proteins from microbial proteolysis in the rumen. Fluorescent cells were easily identified in the presence of strained ruminal fluid when viewed by epifluorescent microscopy, and intact cells were readily enumerated. Batch cultures with rumen digesta demonstrated that 93, 97 and 25% of P. pastoris cells remained intact after 36 to 48 h of incubation in clarified ruminal fluid, an isolated bacterial fraction, and whole ruminal fluid, respectively. In continuous culture (Rusitec) with a dilution rate of 0.75 d-1, 19% of P. pastoris cells flowed intact from the fermentation vessels. In vitro abomasal simulations demonstrated that 84% of inoculated P. pastoris had lysed within 12 h of incubation, a property that is necessary for the release of encapsulated protein prior to the small intestine. These in vitro studies suggest that P. pastoris may be an effective vehicle for post-ruminal delivery of bioactive proteins in ruminants. Key words: Ruminal escape vehicle, Pichia pastoris, green fluorescent protein, bypass protein