Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.     

Dyneins have run their course in plant lineage

Flowering plant genomes lack flagellar and cytoplasmic dyneins as well as the proteins that make up the dynactin complex. The mechanisms for organizing the Golgi apparatus, establishing spindle poles, and moving nuclei, vesicles, and chromosomes in flowering plants must be fundamentally different from those in other systems where these processes are dependent upon dynein and dynactin.     

Interspecific somatic cell hybridization between asparagine-requiring and drug-resistant cell lines

Interspecific hybrids between Walker 256 carcinosarcoma rat cells which are asparagine requiring, and LMTK^t mouse cells which are drug resistant and asparagine independent have been isolated. The hybrids were selectively isolated by taking advantage of the asparagine requirement, or, in some cases, combining the asparagine requirement with an azaguanine resistance marker. The hybrids: (a) possessed a chromosome complement which was additive between the two parent lines; (b) showed two marker chromosomes; (c) possessed both rat and mouse forms of a number of different isozymes. The specific activities of asparagine synthetase was measured in the two parents and the hybrids. The enzyme level in the hybrids was found to be higher than the levels observed in the W 256 line, but only 10% of that observed in the LMTK⁻. The results are in agreement with, but do not prove, the hypothesis that asparagine requirement is due to a mutation in a structural cistron specifying the asparagine synthetase polypeptide.     

Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.     

Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.     

Process development and cGMP manufacturing of a recombinant ricin vaccine: An effective and stable recombinant ricin a‐chain vaccine—RVEc™

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A‐chain (RTA), which is an N‐glycosidase enzyme, and the B‐chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1‐33/44‐198 protein, herein denoted RVEa?) expressed in Escherichia coli using a codon‐optimized gene was shown to be non‐toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale‐up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)‐compliant production of RVEc? at the 40 L scale. The average yield of the final purified bulk RVEc? is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc? was >99% pure by three HPLC methods and SDS‐PAGE. The intact mass and peptide mapping analysis of RVEc? confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc? was immunoprotective against lethal ricin challenge and elicited neutralizing anti‐ricin antibodies in 95–100% of the vaccinated mice. Published 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry

The tissue distribution of folate in its numerous coenzyme forms may influence the development of disease at different sites. For instance, the susceptibility of human colonic mucosa to localized folate deficiency may predispose to the development of colorectal cancer. We report a sensitive and robust ultra high-performance liquid chromatography (UHPLC) tandem mass spectrometry method for quantifying tissue H₄folate, 5-CH₃-H₄folate, 5-CHO-H₄folate, folic acid, and 5,10-CH⁺-H₄folate concentration. Human colonic mucosa (20–100 mg) was extracted using lipase and conjugase enzyme digestion. Rapid separation of analytes was achieved on a UHPLC 1.9-μm C18 column over 7 min. Accurate quantitation was performed using stable isotopically labeled (¹³C₅) internal standards. The instrument response was linear over physiological concentrations of tissue folate (R² > 0.99). Limits of detection and quantitation were less than 20 and 30 fmol on column, respectively, and within- and between-run imprecision values were 6–16%. In colonic mucosal samples from 73 individuals, the average molar distribution of folate coenzymes was 58% 5-CH₃-H₄folate, 20% H₄folate, 18% formyl-H₄folate (sum of 5-CHO-H₄folate and 5,10-CH⁺-H₄folate), and 4% folic acid. This assay would be useful in characterizing folate distribution in human and animal tissues as well as the role of deregulated folate homeostasis on disease pathogenesis.