Molecular Biotechnology. Bernard R. Glick

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Molecular Biotechnology - Bernard R. Glick

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disulfide bonds within a protein. Aberrant disulfide bond formation changes a protein’s configuration, which abolishes protein activity and causes instability. Poor yields of overexpressed proteins often occur because the capacity of the cell to properly fold and secrete proteins has been exceeded.

      Figure 3.29 Summary of protein folding in the endoplasmic reticulum of yeast cells. During synthesis on ribosomes associated with the endoplasmic reticulum (ER), nascent proteins are bound by the chaperones BiP and calnexin, which aid in the correct folding of the protein. Protein disulfide isomerases (PDI) catalyze the formation of disulfide bonds between cysteine amino acids that are nearby in the folded protein. Quality control systems ensure that only correctly folded proteins are released from the ER. Proteins released from the ER are transported to the Golgi apparatus for further processing. Prolonged binding of BiP to misfolded proteins leads to activation of the S. cerevisiae transcription factor Hac1, which controls the expression of several proteins that mediate the unfolded-protein response (UPR). Adapted from Gasser et al., Microb. Cell Fact. 7:11–29, 2008.

      Several strategies have been implemented to increase the host cell’s capacity to process higher than normal levels of proteins. The overproduction of molecular chaperones and protein disulfide isomerases may increase the yield of recombinant proteins, especially those with disulfide bonds. To test this hypothesis, the yeast protein disulfide isomerase gene was cloned between the constitutive glyceraldehyde phosphate dehydrogenase promoter and a transcription terminator sequence in a yeast integrating vector, and the entire construct was integrated into a chromosomal site. The modified strain showed a 16-fold increase in protein disulfide isomerase production compared with the wild-type strain. When protein disulfide isomerase-overproducing cells were transformed with a plasmid vector carrying the gene for human platelet-derived growth factor B, there was a 10-fold increase in the secretion of recombinant protein over that of transformed cells with normal levels of protein disulfide isomerase. The overproduction of protein disulfide isomerase specifically increases the secretion of proteins with disulfide bonds. Higher levels of secreted products were also obtained for the recombinant proteins human erythropoietin, bovine prochymosin, and leech hirudin in S. cerevisiae cells that overexpress the chaperone BiP.

      Overexpression of the molecular chaperone BiP or protein disulfide isomerase increased the secretion of some heterologous protein; however, overexpression of a single chaperone may not have the desired outcome and, in some instances, may increase the degradation of the target protein. This is because proper protein folding requires the coordinated efforts of many interacting factors (Fig. 3.29). Even when levels of one chaperone are adequate, the levels of cochaperones or cofactors may be limiting. The unfolded-protein response of yeast cells coordinates the expression of several chaperones, as well as cochaperones. When the demand for protein folding exceeds the folding capacity of the endoplasmic reticulum, the unfolded-protein response increases the expression of chaperones, protein disulfide isomerase, and other proteins involved in protein secretion. Engineering the proteins of the unfolded-protein response may be a better approach to increase the overall capacity of the cell to fold proteins in a coordinated way by maintaining appropriate ratios of all factors required. Accumulation of unfolded proteins in the endoplasmic reticulum activates the S. cerevisiae transcription factor Hac1, which activates the expression of proteins of the unfolded-protein response, and therefore expression of Hac1 was targeted for genetic manipulation. Overexpression of Hac1 in S. cerevisiae improved secretion of the important industrial enzyme α-amylase, which is used for starch hydrolysis in a wide range of processes.

      Recombinant proteins have been produced successfully in S. cerevisiae from cloned genes from many sources. However, in many cases, expression levels are low and protein yields are modest. One of the major drawbacks of using S. cerevisiae is the tendency for the yeast to hyperglycosylate heterologous proteins by adding 50 to 150 mannose residues in N-linked oligosaccharide side chains that often alter protein function. Although the initial stages of addition of glycan chains to proteins in the lumen of the endoplasmic reticulum are similar in yeast and humans, following transfer of the protein to the Golgi apparatus, further processing differs significantly. The outcome is the production of a sialylated protein in humans and a hypermannosylated protein in yeast, with α-1,3 bonds between the sugar residues that can make the heterologous protein antigenic in humans (Fig. 3.24). Also, proteins that are designed for secretion frequently are retained in the periplasmic space, increasing the time and cost of purification. Finally, S. cerevisiae produces ethanol at high cell densities, which is toxic to the cells and, as a consequence, lowers the quantity of secreted protein. For these reasons, researchers have examined other yeast species and eukaryotic cells that could act as effective host cells for recombinant protein production.

      Pichi pastoris is a methylotrophic yeast that is able to utilize methanol as a source of energy and carbon. It is an attractive host for recombinant protein production because glycosylation occurs to a lesser extent than in S. cerevisiae and the linkages between sugar residues are of the α-1,2 type, which are not allergenic to humans. With these natural characteristics as a starting point, a P. pastoris strain was extensively engineered with the aim of developing a “humanized” strain that glycosylates proteins in a manner identical to that of human cells. Both human and yeast cells add the same small (10-residue), branched oligosaccharide to nascent proteins in the endoplasmic reticulum (Fig. 3.30). However, this is the last common precursor between the two cell types, because once the protein is transported to the Golgi apparatus, further processing is different. In the Golgi apparatus, yeast cells add an α-1,6 mannose residue to the oligosaccharide, which subsequently leads to hypermannosylation. Mammalian cells, on the other hand, remove some mannose residues from the precursor (trimming) and then sequentially add specific sugars to yield a glycoprotein with an oligosaccharide that terminates in sialic acid. To create a “humanized” strain, the enzyme responsible for addition of the α-1,6 mannose was first eliminated from P. pastoris to prevent hypermannosylation. Next, the gene encoding a mannose-trimming enzyme (a mannosidase) from the filamentous fungus Trichoderma reesei was inserted into the yeast genome and was found to trim the oligosaccharide to a human-like precursor. Genes encoding enzymes for the sequential addition of sugar residues that terminate the oligosaccharide chains in galactose were also added. It should be noted that the coding sequences for all engineered genes contained a secretion signal for localization of the encoded protein to the Golgi apparatus. Finally, several genes for proteins that catalyze the synthesis, transport to the Golgi apparatus, and addition of sialic acid to the terminal galactose on the protein precursor were inserted into the P. pastoris genome. Properly sialylated recombinant proteins, including erythropoietin and antibodies, that can be used as human therapeutic agents have been produced by the “humanized” P. pastoris.

      Figure 3.30 Differential processing of glycoproteins in P. pastoris, humans, and “humanized” P. pastoris. Initial additions of sugar residues to glycoproteins in the endoplasmic reticulum are similar in human and P. pastoris cells (A). However, further N-glycosylation in the Golgi apparatus differs significantly between the two cell types. N-glycans are hypermannosylated in P. pastoris (B), while in humans, mannose residues are trimmed and specific sugars are added, leading to termination of the oligosaccharide in sialic acid (C). P. pastoris cells have been engineered to produce enzymes that process glycoproteins in a manner similar to that of human cells. In “humanized” P. pastoris, a recombinant glycoprotein produced in the endoplasmic reticulum (D) is transported to the Golgi apparatus, where it is further processed to yield a properly sialylated glycoprotein (E). Blue squares, N-acetylglucosamine; red circles, mannose; green squares, galactose; orange squares, sialic acid. Adapted from Hamilton and Gerngross, Curr. Opin. Biotechnol. 18:387–392,

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