Yeast proteins amino acid sequencing

Using rational metabolic engineering to tailor the stereoselectivity of yeast-mediated reductions is feasible only when all of the relevant genes are known and the relationships between enzyme and acceptable substrates are defined. We are far from this ideal. For a few reductase proteins, amino acid sequence data has... 

The leucine zipper motif (see Chapter 3) was first recognized in the amino acid sequences of a yeast transcription factor GCN4, the mammalian transcription factor C/EBP, and three oncogene products, Fos, Jun and Myc, which also act as transcription factors. When the sequences of these proteins are plotted on a helical wheel, a remarkable pattern of leucine residues... 

Consensus sequences similar to ori or ARS in structure or function have not been precisely defined in mammalian cells, though several of the proteins that participate in ori recognition and function have been identified and appear quite similar to their yeast counterparts in both amino acid sequence and function. 

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. 

Of these, 11 function with ubiquitin and one with each of two different ubiquitin-like (UbL) proteins. Sumo and Nedd8/Rubl [2]. However, while several yeast and metazoan E3s had been identified biochemically by that time, the primary amino acid sequence was known only for a single E3 [7]. This was the Saccharomyces cer-evisiae Ubrlp, which had been studied extensively by Varshavsky and co-workers in delineation of the N-end rule [8, 9]. Its mammalian ortholog, E3a, was the prototypical E3 used in the description of the basics of the ubiquitin pathway by Hershko and Ciechanover [1, 10]. 

Some human proteins produced in a bacterium will not work even if the amino acid sequence is identical to the human protein. This is because our cells, and the cells of plants, animals, and yeast, add sugars to most of the proteins they produce. There are many different kinds of sugar molecules, and the type of sugar added and the place in the cell and on the protein where the sugars are added vary among cells for different species. If the correct sugars are not on the correct spot on the protein, the protein may not fit into the receptor pocket correctly, and thus not have the desired effect. Also, without the added sugars, the enzymes in our blood may destroy the protein before it can get to the appropriate spot to bind to the correct receptor. 

Half-lives were measured in yeast for the j8-galactosidase protein modified so that in each experiment it had a different amino-terminal residue. (See Chapter 9 for a discussion of techniques used to engineer proteins with altered amino acid sequences.) Half-lives may vary for different proteins and in different organisms, but this general pattern appears to hold for all organisms. 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

A hard-to-understand aspect of the "protein-only" theory of prion diseases is the existence of various "strains" of prion proteins. These do not involve differences in amino acid sequence but differences in the conformations of the PrPSc forms and in the glycosylation patterns. dmw How can there be several different conformations of the same protein, all of which seed the conversion of normal PrP into differing insoluble forms In spite of this puzzle, support for the explanation of strain differences comes from a yeast prion system, which involves transcription termination factor eRF3.x z In this system, which involves a prion whose insoluble form can be redissolved by guanidine hydrochloride,aa differing strains have also been described.ybb cc Nevertheless, the presence of the various strains of animal prions, as well as observed vaccination of inbred mice against specific strains,dd may be more readily understood if the disease is transmitted by an unidentified virus rather than by a pure protein.1/U ee/ff In fact, the diseases have not been successfully transmitted by truly virus-free proteins synthesized from recombinant DNA.ee... 

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