Yeast recombinant proteins produced

Mattanovich D, Gasser B, Hohenblum H et al (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113 121-135... 

We have experimented with Trp analog labeling protocols for recombinant proteins produced in bacteria and yeast, and we have found variability in these labeled proteins both with respect to structural and functional integrity and degree of analog incorporation (1,2). We have evaluated levels of 5-OHTrp incorporation in several protein systems. In this paper, we evaluate attempts to replace the four Trps in the truncated, soluble domain of recombinant human Tissue Factor (sTF) with 5-OHTrp and 7-ATrp. This domain of native, membrane-bound tissue factor binds factor Wa, a serine protease circulating in... 

Topical anti-ulcer. Recombinant protein produced in the yeast, Saccharomyces cerevisiae. Homodimer. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

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. 

Differences in post-translational modification (PTM) detail. Human therapeutic proteins produced in several recombinant systems (e.g. yeast-, plant- and insect-based systems Chapter 5) can display altered PTM detail, particularly in the context of glycosylation (Chapter 2). Some sugar residues/motifs characteristic of these systems can be highly immunogenic in humans. 

Yeast expression vectors have been among those most commonly used since the beginning of gene technology. Vectors based on baker s yeast, Saccharomyces cerevisiae, have been especially popular for robust expression of many types of recombinant proteins [90]. For instance, the first commercially available recombinant vaccine, the hepatitis B surface antigen vaccine, was produced from an S. cerevisiae vector [91]. Many other recombinant proteins have also been efficiently expressed in yeast including al-Antitrypsin [92], insulin [93], Epstein-Barr virus envelope protein [94], superoxide dismutase [95] and interferon-a [90]. 

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... 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

Historically, HCDC was first established for yeasts to produce single-ceU protein, ethanol, and biomass. Later, dense cultures of other mesophiles producing various types of products were developed, e.g.,by Suzuki et al. [96]. The combination of recombinant DNA technology and large-scale culture processes has enabled human proteins to be produced in a number of hosts, in particular in Escherichia coli [97-100]. Approaches to optimize the production of recombinant proteins are the subject of recent reviews from Winter et al. [101]. 

Feedstocks of yeast cell suspensions are relatively common starting material, as the yeast cell usually exports the expressed recombinant protein efficiently in the culture medium. A process was developed for the recovery of human serum albumin produced in Pichia pastoris using a 5 cm diameter column run in expanded-bed mode and scaled up directly to a 100 cm diameter column using 150 liters of cationic exchangers. Using this size of column, it was possible to apply a 1900 liter cell suspension. The yield of human serum albumin varied from 83 to 91% over four different runs.22... 

Currently, 60 to 70 percent of all biotherapeutics based on recombinant proteins are produced in cultivated mammalian cells.8 Mammalian systems are often preferred over other hosts, such as bacteria, plants, and yeast, because of their capability for proper protein folding, assembly, and posttranslational modification. The quality and efficacy of a protein can thus be enhanced when expressed in mammalian cells. With the recent expansion in the biotherapeutics market, the demand for proteins derived from mammalian cells... 

For the production of many recombinant proteins, cellular insolubility is a major problem because it is often difficult to achieve biological activity starting with such products (57). However, because the polyphenolic protein produced in vivo is a preadhesive, in vitro activation to the adhesive form is required. Therefore, in vivo insolubility may actually be desirable in the case of the polyphenolic protein because this could result in increased resistance to yeast proteases and better product uniformity and quantity. 

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