The flasks were subsequently incubated at 25C around the orbital shaker (120 rpm) under dark/light conditions as specified by individual treatments. and the antibiotic marker-free transformation system offers a novel strategy to achieve high yields of complex therapeutic proteins secreted from herb roots. Therapeutic recombinant proteins have been produced in many different hosts, both prokaryotic and eukaryotic (Fischer et al., 1999). Each of them provides a unique set of advantages and can be tailored to the production of a target protein, depending on the specific requirements imposed by the manufacturing process. When the protein of interest originates from a eukaryotic source, the manufacturing method of choice primarily depends on the yield, codon usage, solubility, and set of complex posttranslational modifications required for structural integrity and biological Tpo activity of the Climbazole protein (Higgins and Hames, 1999). Most first-generation recombinant proteins were well-characterized peptides, such as insulin and other hormones, which functioned as therapeutic agents just as they normally would (Gibbons, 1991). Many second- and third-generation recombinant products, however, are complex monoclonal antibodies (mAbs) that require multiple processing actions to preserve their initial bioactivity. Therefore, high costs and limited production capacities remain the major obstacles to many long-term therapies based on mAb treatments (Maloney et al., 1997). In general, plant-based systems compare favorably with option expression platforms, both in terms of quality and cost of complex therapeutic proteins. After a routine transformation protocol was developed for plants, two research groups successfully expressed full-size recombinant antibodies in tobacco (Nicotiana tabacum) leaf tissue (During, 1988;Hiatt et al., 1989). Since then, a variety of antibody fragments and/or full-length Climbazole mAbs have been produced in plants (Stoger et al., 2002). Biologically active mAbs Climbazole require a number of assembly actions and posttranslational modifications that are carried out in the endoplasmic reticulum (ER). Once the recombinant protein is directed to the ER, it is generally secreted to the apoplast following the default secretion pathway (Deneke et al., 1990), targeted to the vacuole (Frigerio et al., 2002), or retained in the ER by the addition of the KDEL C-terminal sequence (Conrad and Fiedler, 1998). Proteases released during herb tissue harvesting, extraction, and downstream protein purification often result in antibody degradation (Ma et al., 1994;Sharp and Doran, 2001). Using the nondestructive secretion process that provides high yields of recombinant proteins over the lifetime of a herb and facilitates downstream purification can circumvent this manufacturing challenge. Two related herb production systems have been designed recently to achieve a nondestructive production process utilizing rhizosecretion (Borisjuk et al., 1999) or guttation (Komarnytsky et al., 2000). The rhizosecretion of a functional murine mAb from the roots of previously transformed tobacco plants, resulting in a mean antibody yield of 12g/g root Climbazole dry weight per day, was exhibited subsequently (Drake et al., 2003). Here, we describe an optimized antibiotic-free transformation and rhizosecretion system for stable high-yield production of complex proteins based on the pRYG transformation vector (Komarnytsky et al., 2004). The system was engineered to provide enhanced levels of tissue-specific expression of the human single-chain IgG1and full-length IgG4immunoglobulin complexes and improve production rates based on the use of plant-derived signal peptides. Additionally, we demonstrate that cosecretion of the Bowman-Birk Ser protease inhibitor (BBI) into the herb growth medium significantly enhances antibody stability and yield. == RESULTS AND DISCUSSION == Speed of development, as well as increasing stability and yield of the target protein, are the most important factors if plants are to become a system for the commercial manufacturing of therapeutic recombinant proteins (Peeters et al., 2001). The utilization of the pRYG transformation vector harboring a cluster ofrolgenes is usually a fast and effective method for generating transgenic plants without the introduction of antibiotic resistance (Komarnytsky et al., 2004). This vector rapidly induces a large number of independently transformed adventitious roots, enabling efficient screening of individual root clones, selection of the best suppliers, and subsequent regeneration of fertile plants from them (Gaume et al., 2003). To estimate the efficiency and production capacity of the system, we attempted a rhizosecretion of both single-chain and full-length human mAbs. == Selection of Genetic Elements for Single-Chain IgG1Production == To fully capitalize on rhizosecretion capacity, we have used an amplification-promoting sequence (aps; known to stabilize.