Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

Andrea Kerekes,Nándor Lipták,Elen Gócza,László Hiripi,Wilfried A Kues,Zsuzsanna Bősze,Orsolya Ivett Hoffmann,Gergely Iski,Bin He

doi:10.1371/journal.pone.0187214

Abstract

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

Highlights

Sheep β-lactoglobulin [1] and human tissue plasminogen activator [2] were the first proteins produced in the milk of transgenic mice in 1987
We demonstrated the expression of the Venus recombinant protein driven by the ubiquitous CAG promoter in milk and other biological fluids of transposon-carrying transgenic rabbits despite the absence of a signal peptide sequence for the secretory pathway
Venus protein was detected by Western blot analysis in all milk fractions of the Sleeping Beauty (SB)-CAG-Venus transgenic does during a lactation period

Summary

Introduction

Sheep β-lactoglobulin [1] and human tissue plasminogen activator [2] were the first proteins produced in the milk of transgenic mice in 1987. Recombinant proteins may be sourced from milk, but from blood, egg white, and seminal plasma. Purifying recombinant proteins from milk has several advantages: milk is collectable from lactating animals, tremendous amounts of proteins can be harvested, and milk contains only small amounts of proteases. Bioactive proteins in the blood of transgenic animals may have adverse effects on the animals’ health, and blood samples are more frequently contaminated with pathogens (review [3]).

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