Plant-based production of xenogenic proteins.

Foreign protein production in transgenic plants has been successful, from the generation of transgenic plant lines to the marketing of purified proteins. Antigenic proteins from disease organisms, monoclonal antibodies raised against antigens of disease organisms, and proteins with industrial process applications have been produced and tested. For vaccines, clinical trials in humans and feeding trials in animals are in progress to demonstrate their efficacy. For industrial proteins, high expression and downstream processing efficiency are key concerns, with application and test market trials in progress.

Differentiation of potyviruses and their strains by hybridization with the 3' non-coding region of the viral genome.

Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.

Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: an example of beta-glucuronidase.

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.

Mechanism of activation of bovine kidney pyruvate dehydrogenase a kinase by malonyl-CoA and enzyme-catalyzed decarboxylation of malonyl-CoA.

The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.

Properties of a newly characterized protein of the bovine kidney pyruvate dehydrogenase complex.

The dihydrolipoyl transacetylase component, which serves as the structural core of mammalian pyruvate dehydrogenase complexes, is acetylated when treated with either pyruvate or with acetyl-CoA in the presence of NADH. Besides the dihydrolipoyl transacetylase component, we have found that another protein, referred to as protein X, is rapidly acetylated at thiol residues. Protein X remains fully bound to the transacetylase core under conditions that remove the pyruvate dehydrogenase and dihydrolipoyl dehydrogenase components. Mapping of 125I-tryptic peptides indicated that the transacetylase subunits and protein X are structurally distinct; however, under the same mapping conditions, there is considerable similarity in the positions of acetylated peptides derived from these subunits. Affinity-purified rabbit immunoglobulin G prepared against the dihydrolipoyl transacetylase core reacted exclusively with the transacetylase and with both its tryptic-derived inner domain and outer lipolyl-bearing domain. Those results further indicate that protein X is not derived from the transacetylase subunit Affinity-purified mouse antibody to protein X reacted selectively with large tryptic polypeptides derived from protein X and did not react with the inner domain of the transacetylase. However, the anti-protein X antibody did react with the intact transacetylase subunit, the lipoyl-bearing domain of the transacetylase, and weakly with the transsuccinylase component of the alpha-ketoglutarate dehydrogenase complex. This cross-reactivity reflected specificity of a portion of the polyclonal antibodies for a related structural region in the transacetylase and protein X (possibly a similar lipoyl-bearing region). Furthermore, a major portion of that polyclonal antibody was shown to react exclusively with protein X. Thus, protein X subunits differ substantially from transacetylase subunits but the two components have a region of structural similarity. We estimate that there are about 5 mol of protein X per mol of the kidney pyruvate dehydrogenase complex. Under a variety of conditions that result in a wide range of levels of acetylation of sites in the complex, about 1 acetyl group is incorporated into protein X per 10 acetyl groups incorporated into the transacetylase subunits per mol of complex. That ratio is close to the ratio of protein X subunits of transacetylase subunits in the complex, indicating that there are efficient mechanisms for acylation and deacylation of protein X.

Properties of the pyruvate dehydrogenase kinase bound to and separated from the dihydrolipoyl transacetylase-protein X subcomplex and evidence for binding of the kinase to protein X.

Studies were conducted on four pyruvate dehydrogenase kinase-containing fractions: purified pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2.X.K), a kinase fraction (K fraction) prepared from the E2.X.K subcomplex, and a kinase fraction generated by limited trypsin-digestion of E2.X.K. We characterized the gel electrophoresis properties of dissociated subunits (one-dimensional and two-dimensional), the catalytic and ATP binding properties of kinase-containing fractions, and the subunit requirements for kinase binding to and being activated by the transacetylase-protein X subcomplex (E2.X). A significant portion of protein X was retained with the transacetylase core following release of virtually all the kinase. The K fraction had four major bands separated by sodium dodecyl sulfate-slab gel electrophoresis which corresponded to the dihydrolipoyl dehydrogenase, protein X, the trypsin-resistant catalytic subunit of the kinase and a chymotrypsin-resistant subunit which had a high pI and comigrated in one-dimensional systems with the chymotrypsin-sensitive alpha-subunit of the pyruvate dehydrogenase component. While purified kidney complex contained only about three molecules of kinase (determined by [14C]ATP binding), one molecule of E2.X subcomplex activated a large number (greater than 15) molecules of kinase associated with the protein X-containing K fraction. Sephadex G-200 chromatography of the K fraction in the presence of dithiothreitol led to coelution of protein X and kinase subunits. Limited trypsin digestion converted the transacetylase into subdomains and cleaved protein X and the high pI subunit of the kinase. Under those conditions, the intact catalytic subunit of the kinase did not bind to the large inner domain of the transacetylase but could be activated by untreated E2.X subcomplex. Thus, binding of the catalytic subunit of the kinase and its activation by E2.X required either protein X or the lipoyl-bearing outer domain of the transacetylase. In combination, our results suggest that protein X serves to anchor the kinase to the core of the complex.

Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus.

The sequence of the 3'-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3' terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3' non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3' non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3' non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.

Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.