Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture.
The heterologous protein expression system of Pichia pastoris is now widely used for expression of many human proteins, because the efficiently expressed proteins will be correctly folded in Pichia pastoris cells and also efficiently secreted from the cells. Recombinant human serum albumin (rHSA) is efficiently secreted from Pichia pastoris. Nowadays, the expression of rHSA exceeds 10g in 1 L fermentor culture broth, and the protein is completely purified. Recombinant HSA expressed in Pichia pastoris was approved as a medicine by the authorities in 2007, and launched in 2008 in Japan. One of the insulin precursors (IP) was also successfully expressed in Pichia pastoris, and secreted up to 3.6g in 1 L medium using a multi-copy transformant. The insulin precursor could be efficiently converted to insulin, the final product, in vitro. Human growth hormone was also expressed in Pichia pastoris, and secreted up to 49 mg in 1 L medium. These proteins are also important for clinical applications. Midkine and pleiotrophin may be two of the candidates for clinical applications. Secretion signals, the copy number of an expression cassette in transformants, and culture conditions for fermentation were examined for efficient expression of these proteins in Pichia pastoris. The best signal was selected, and other factors were optimized. The amounts of native midkine and native pleiotrophin expressed were approximately 0.36g and 0.26g in 1 L medium, respectively. Expression of bile-salt stimulated lipase (BSSL) had been extremely low in the beginning of a fermentor culture experiment. However, approximately 1 g rBSSL in 1 L medium was finally expressed in a fermentor by unlimited feeding of glycerol for cell growth and optimization of other factors. BSSL from human milk and rBSSL from Pichia cells are glycosylated. The structure differences between these glycans are obvious. When humanization of Pichia glycans is established by genetic engineering, the Pichia pastoris expression system will become indispensable for the production of therapeutic proteins.
Murasugi
Akira
A
Product Development Department, Meiji Dairies Corporation, 1-2-10, Shin-suna, Koto-ku, Tokyo 136-8908, Japan. akira_murasugi@meiji-milk.com
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D005285:Fermentation; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins