A chloroplast transgenic approach to hyper‐express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation

Abstract

SummaryHuman Serum Albumin (HSA) accounts for 60% of the total protein in blood serum and it is the most widely used intravenous protein in a number of human therapies. HSA, however, is currently extracted only from blood because of a lack of commercially feasible recombinant expression systems. HSA is highly susceptible to proteolytic degradation in recombinant systems and is expensive to purify. Expression of HSA in transgenic chloroplasts using Shine-Dalgarno sequence (SD), which usually facilitates hyper-expression of transgenes, resulted only in 0.02% HSA in total protein (tp). Modification of HSA regulatory sequences using chloroplast untranslated regions (UTRs) resulted in hyper-expression of HSA (up to 11.1% tp), compensating for excessive proteolytic degradation. This is the highest expression of a pharmaceutical protein in transgenic plants and 500-fold greater than previous reports on HSA expression in transgenic leaves. Electron micrographs of immunogold labelled transgenic chloroplasts revealed HSA inclusion bodies, which provided a simple method for purification from other cellular proteins. HSA inclusion bodies could be readily solubilized to obtain a monomeric form using appropriate reagents. The regulatory elements used in this study should serve as a model system for enhancing expression of foreign proteins that are highly susceptible to proteolytic degradation and provide advantages in purification, when inclusion bodies are formed.

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

“…The chloroplast transformation vector pLDK, which contains the chorismate pyruvate-lyase gene (ubiC), was expressed under the control of the psbA 5# untranslated region (UTR)/promoter to maximize expression. It has previously been shown that genes under the control of the psbA promoter/5#UTR achieve very high levels of expression (Fernandez San-Millan et al, 2003;Dhingra et al, 2004;Watson et al, 2004). The 5#UTR has been hypothesized to enhance translation of proteins under its control (Eibl et al, 1999).…”

“…Because of these advantages the chloroplast genome has been engineered to confer several useful agronomic traits, including herbicide resistance (Daniell et al, 1998), insect resistance (McBride et al, 1995;Kota et al, 1999), disease resistance (DeGray et al, 2001), drought tolerance (Lee et al, 2003), salt tolerance , and phytoremediation (Ruiz et al, 2003). The chloroplast genome has also been used in molecular farming to express human therapeutic proteins (Guda et al, 2000;Staub et al, 2000;Fernandez-San Millan et al, 2003;Daniell et al, 2004bDaniell et al, , 2004c, vaccines for human (Daniell et al, 2001a(Daniell et al, , 2004cDaniell, 2004;Watson et al, 2004) or animal use (Molina et al, 2004), and biomaterials (Guda et al, 2000;Lossl et al, 2003). Although most of these studies were done in tobacco, highly efficient stable plastid transformation of major crop species has recently been reported for carrot , cotton (Gossypium hirsutum; Kumar et al, 2004b), and soybean (Glycine max; Dufourmantel et al, 2004).…”

Large-scale production of functional human serum albumin from transgenic rice seeds

“…Over the past decades, various expression systems have been used to produce rHSA, including Escherichia coli (13), Saccharomyces cerevisiae (14), Kluyveromyces lactis (15), Pichia pastoris (16), transgenic animals (17), and transgenic plants (18)(19)(20)(21). Attempts to produce rHSA in tobacco leaves and potato tubers achieved expression levels of 0.02% of total soluble protein (TSP) (18), and expression was increased to 0.2% of TSP by targeting the rHSA to the apoplast of potato tubers (19).…”

“…Attempts to produce rHSA in tobacco leaves and potato tubers achieved expression levels of 0.02% of total soluble protein (TSP) (18), and expression was increased to 0.2% of TSP by targeting the rHSA to the apoplast of potato tubers (19). Recently, an expression level of 11.1% of TSP was obtained by expressing rHSA in tobacco leaf chloroplasts (20). More recently, an rHSA expression level of 11.5% of total proteins was achieved in a rice cell culture by a sugar starvation-induced promoter (21).…”

Chloroplast Genetic Engineering

“…In addition to its use for the hyper-expression of vaccine antigens, the tobacco chloroplast has been used in the Daniell laboratory for production of valuable therapeutic proteins, such as human elastin-derived polymers for various biomedical applications [25], human serum albumin [26], magainin, a broad spectrum topical agent, systemic antibiotic, wound healing stimulant and a potential anticancer agent [18], interferon and insulin-like growth factor [3,6]. These examples show that chloroplast also contains the machinery that allows for correct folding and disulfide bond formation, resulting in fully functional proteins.…”