Secreted protein purification

LSBC has a long history of process development, scale-up and manufacturing experience, since its foundation back in 1987. The company has developed proprietary, industrial-scale extraction processes for the purification of proteins, peptides, and other biochemicals from plant biomass. It has also developed commercial methods for the extraction and purification of secreted plant proteins. 

Secreted proteins from eukaryotic cell lines present problems in purification due to both the volume of... 

In summary, advances in our understanding of the nutritional and hormonal requirements of cells in culture and of the role played by attachment factors and transport proteins have led to the possibility that any cell can be cultured in the absence of serum. The type of serum substitute used and the means of achieving a serum-free culture system should be determined by the ultimate purpose that the culture system is to serve. The advantages of serum-free culture are manifest both in the practical realm of providing an inexpensive and simple starting material for the purification of cell-secreted proteins, such as antibodies, and for providing a more precise definition of the in vitro environment in order to model more... 

The P. past oris expression system produced high yields of secreted protein. The recombinant protein represents the majority of the protein supernatant without further purification. The E. coli expression system was found to be unsuitable for expression of soluble and active ferulic acid esterase from a fungal source. 

Fetal bovine serum (FBS) (v/p) is added to the medium of SF900 II SFM for Sf9 propagation, transfection, virus amplification and intracellular expression. FBS is omitted from the medium for extracellular expression of secreted protein. The presence of FBS in the medium may mask the expression of the secreted target protein and may interfere with the purification procedure. 

The costs of purification of protein polymers can be high if purification requires multiple chromatographic separations however, most repetitive protein poljuners have physicochemical properties very different from cellular proteins, and can be separated from these proteins by selective precipitation methods involving changes in temperature and pH. Similarly, protein polymers whose sequences alone may not permit such simple purification strategies can be expressed with fusion tags that facilitate purification. Secretion into the culture medium can also simplify purification and provide opportunities for less complicated large-scale syntheses, as expression hosts could be immobilized on a solid support, and protein removed in a continuous fashion without the requirement to turn over the culture entirely. 

Another well established immobilization technique is via recombinant tags like polyhistidine tags. The idea came from immobilized metal affinity chromatography (IMAC) for the purification of proteins tagged with polyhistidine [145]. This method allows immobilization without prepurification steps and shows no interference with the enzyme structure or function, and does not affect the secretion or folding of fusion proteins within cells [146]. 

Yeast. The advantages of expression in yeast include potentially high level production of proteins, the abiUty to have expressed proteins secreted into the media for ease of purification, and relatively low cost, easy scale-up. A disadvantage is that plasmid instabiUty may be a problem which can lead to low product yield. Whereas post-translational modification occurs in yeast, proteins are quite often hyperglycosylated. This is generally a problem with expression in Saccharomyces cerevisiae but not for the more recently used yeast host Pichiapastoris (25) (see Yeasts). 

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