Production of a recombinant industrial protein using barley cell cultures.

The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.

Vacuolar sorting determinants within a plant storage protein trimer act cumulatively.

The mechanism for vacuolar sorting of seed storage proteins is as yet poorly understood and no receptor has been identified to date. The homotrimeric glycoprotein phaseolin, which is the major storage protein of the common bean, requires a transient tetrapeptide at the C-terminus for its vacuolar sorting. A mutated construct without the tetrapeptide is secreted. We show here that coexpression of wild-type phaseolin and the mutated, secreted form in transgenic tobacco results in the formation of mixed trimers and partial vacuolar delivery of the mutated polypeptides and partial secretion of wild-type polypeptides. This indicates that the sorting signal has a cumulative effect within a phaseolin trimer. The result is discussed in the light of the hypothesized mechanisms for vacuolar sorting of seed storage proteins.

Different degradation pathways for heterologous glycoproteins in yeast.

Rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli beta-lactamase were translocated into the yeast endoplasmic reticulum (ER), glycosylated, misfolded and rapidly degraded. NGFRe underwent ATP-dependent thermosensitive degradation independently of vesicular transport. Since no evidence for degradation by the cytoplasmic 26S proteosome complex could be obtained, NGFRe appeared to be degraded in the ER. Beta-lactamase exited the ER by vesicular traffic and was transported from the Golgi via the Vps10 receptor pathway to the vacuole for degradation. Machineries in the ER and the Golgi appear to recognize distinct structural features on misfolded heterologous proteins and guide them to different degradation pathways.

Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo.

We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent. Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.

The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates.

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.

Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae.

Here we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp 150 delta) carrier. The hsp 150 delta-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFRe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp 150 delta-NGFRe fusion protein were highly selectively O-glycosylated.

Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast.

Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.