A Chloroplast Transgenic Approach to Hyper-Express and Purify Human Serum Albumin, a Protein Highly Susceptible to Proteolytic Degradation

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Departmental Papers (Dental)
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chloroplast genetic engineering
biopharmaceuticals
genetically modified crops
molecular farming
recombinant human blood proteins
Dentistry
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Millán, Alicia Fernández-San
Mingo-Castel, Angel
Miller, Michael
Daniell, Henry
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Human 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.

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2003-03-01
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Plant Biotechnology Journal
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At the time of publication, author Henry Daniell was affiliated with the University of Central Florida. Currently, he is a faculty member at the School of Dental Medicine at the University of Pennsylvania.
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