Yeast cells, recombinant proteins

Steinborn, G., Boer, E., Scholz, A. et al. (2006) Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microbial Cell Factories, 5, 33. 

Many of the initial biopharmaceuticals approved were simple replacement proteins (e.g. blood factors and human insulin). The ability to alter the amino acid sequence of a protein logically coupled to an increased understanding of the relationship between protein structure and function (Chapters 2 and 3) has facilitated the more recent introduction of several engineered therapeutic proteins (Table 1.3). Thus far, the vast majority of approved recombinant proteins have been produced in the bacterium E. coli, the yeast S. cerevisiae or in animal cell lines (most notably Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. These production systems are discussed in Chapter 5. 

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... 

In addition to the limited number of expressed genes, bacteria feature a highly reproductive cycle but no cell nucleus, no organelles for compartmentation, no cell-to-cell communication as in tissues, and quite a straightforward metabolism. Favored bacterial systems for production of recombinant proteins are various strains of E. coli, Bacillus, and Pseudomonas. Non-bacterial options such as yeast or mammalian cells are discussed briefly later in this chapter. 

The A. saitoi carboxypeptidase cDNA was cloned downstream of a GDP promoter, and the resulting plasmid, pGCP13, was used to generate recombinant A. saitoi carboxypeptidase protein. No enzymatic activity was detected in the culture supernatant. We detected the A. saitoi carboxypeptidase activity of the extract obtained from yeast cells transfected with A. saitoi carboxypeptidase cDNA in forward orientation (pGCP13), although no activity was observed with the vector alone at pH 3.1. The recombinant A. saitoi carboxypeptidase activity... 

When using common E. coli expression system, recombinant allergens are present, either soluble in the supernatant of the cell lysate, or the proteins aggregate and form poorly soluble inclusion bodies. Inclusion bodies have to be solubilized under denaturing conditions (e.g., urea, guanidine-HCl treatment) and the protein has to be carefully refolded (Rea et al. 2004). One advantage of a eukaryotic yeast expression system is that yeast cells can segregate the expressed protein into the culture medium and thus can easily be purified. 

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