Production of recombinant human serum albumin in transgenic plants and plant cells

摘要

Human serum albumin (HSA) is the most abundant protein in the circulatory system. The principal function of HSA is to transport fatty acids, but it is also capable of binding a great variety of metabolites and drugs. The potential of HSA in various applications, like usage as a plasma expander, stabilizing agent, drug delivery protein and as an adjunct in insulin action prolongation demands a great supply of pure HSA. Presently HSA is manufactured from the blood pooled from commercial blood donors. There is no guarantee regarding contamination of the product by viral pathogens and prions, causing dreadful diseases. The annual world need exceeds 500 tons, representing a market value of more than 1.5 billion US$ (Fernandez-San Millan et al. 2003). Considering the amount of plasma expanders required in critically ill patients, the use of albumin represents a significant cost (Nicholson et al. 2000). Such a huge demand can not longer be met by merely purifying HSA from original sources, as the supply of human blood is limited. Alternative ways to obtain HSA are highly attractive both to decrease the dependence on blood banks and to reduce possible health hazards, such as AIDS and hepatitis that may be transmitted during/by infusion of products derived of contaminated blood. The initial attempt was to generate a novel plant codon optimised semi-synthetic HSA gene by PCR amplification. The strategy was to eliminate the rare codons that are used at low frequencies in the plant host system resulting in an optimized codon usage pattern for high-level expression of recombinant HSA (rHSA). Sequence analysis revealed the presence of high number mutations in the final product, including insertions and deletions of several bases which could not be eliminated. The main focus of the work was to establish an alternative expression system for the production of the authentic HSA cDNA. Further analysis included expression and characterization of rHSA in E. coli, transient expression in leaves of tobacco, zucchini and bean, stable expression in (intact) tobacco and wheat plants and tobacco cell suspension culture. For bacterial expression of rHSA, cytoplasmic and periplasmic expression vectors were generated. After verifying expression in E. coli, purification of the recombinant protein was carried out. Tobacco, zucchini and bean host systems were assessed for expression of rHSA. No difference in transient accumulation levels were observed when the protein was either secreted to the apoplast or targeted to endoplasmic reticulum (ER). Further analysis included stable transformation of tobacco plants via Agrobacterium mediated gene transfer. Accumulation of rHSA in the ER and secretion into the apoplast were confirmed by immunoblot analysis. In parallel to the expression studies in intact plants, tobacco suspension cultures were initiated and both intracellular and secreted protein could be detected in batch culture of the transgenic BY2 cells. Protein stability following secretion could be improved with the addition of appropriate chemical agents (stabilizers) to the growth media. The results achieved by all expression systems and their comparison were performed. Finally downstream processing of the rHSA from transgenic tobacco leaves was investigated. Conditions for an efficient extraction of the recombinant protein from plant system were optimised and cation exchange chromatography to partially purify rHSA was carried out.