Metabolic profiling of amino acids in cellular samples via zwitterionic sub-2 µm particle size HILIC-MS/MS and a uniformly 13C labeled internal standard.

A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-µm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly (13)C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % (n = 5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography-tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).

Measurement uncertainty of isotopologue fractions in fluxomics determined via mass spectrometry.

Metabolic flux analysis implies mass isotopomer distribution analysis and determination of mass isotopologue fractions (IFs) of proteinogenic amino acids of cell cultures. In this work, for the first time, this type of analysis is comprehensively investigated in terms of measurement uncertainty by calculating and comparing budgets for different mass spectrometric techniques. The calculations addressed amino acids of Pichia pastoris grown on 10% uniformly (13)C labeled glucose. Typically, such experiments revealed an enrichment of (13)C by at least one order of magnitude in all proteinogenic amino acids. Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The samples were diluted to fit the linear dynamic range of the mass spectrometers used (10 µM amino acid concentration). The total combined uncertainties of IFs as well as the major uncertainty contributions affecting the IFs were determined for phenylalanine, which was selected as exemplary model compound. A bottom-up uncertainty propagation was performed according to Quantifying Uncertainty in Analytical Measurement and using the Monte Carlo method by considering all factors leading to an IF, i.e., the process of measurement and the addition of (13)C-glucose. Excellent relative expanded uncertainties (k = 1) of 0.32, 0.75, and 0.96% were obtained for an IF value of 0.7 by LC-MS/MS, GC-MS, and LC-TOFMS, respectively. The major source of uncertainty, with a relative contribution of 20-80% of the total uncertainty, was attributed to the signal intensity (absolute counts) uncertainty calculated according to Poisson counting statistics, regardless which of the mass spectrometry platforms was used. Uncertainty due to measurement repeatability was of importance in LC-MS/MS, showing a relative contribution up to 47% of the total uncertainty, whereas for GC-MS and LC-TOFMS the average contribution was lower (30 and 15%, respectively). Moreover, the IF actually present also depends on the isotopic purity of the carbon sources. Therefore, in the uncertainty calculation a carbon source purity factor was introduced and a minor contribution to the total uncertainty was observed. The results obtained by uncertainty calculation performed according to the Monte Carlo method were in agreement with the uncertainty value of the Kragten approach and showed a Gaussian distribution.

Flow cytometric analysis of metabolic stress effects due to recombinant plasmids and proteins in Escherichia coli production strains.

Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells. Using flow cytometry, we analyzed the temporal development of the cellular content of DNA, total protein, and the recombinant product (human superoxide dismutase) in different strains. In cells carrying plasmids utilizing the phage T7 promoter 10 (pET vectors), induction with IPTG leads to an increase in protein content and size, an increase and a wide spreading of DNA content distribution, and a termination of cell division. These effects occurred with pET plasmids with or without an insert, but not with another plasmid which utilizes the tac promoter.

Recombinant expression of alliin lyase from garlic (Allium sativum) in bacteria and yeasts.

Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.

Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells.

The production of foreign proteins at high yields represents a severe metabolic stress for Escherichia coli cells. In many cases, induction of protein synthesis results in rapid exhaustion of the cellular energy and metabolic precursors and thus in cell death. Therefore, sustained production of foreign proteins requires some fine tuning of the specific production rate to meet the capabilities of the cell. This has stimulated us to analyze by flow cytometry the physiological behaviour of recombinant E. coli cells producing human superoxide dismutase (SOD). Two strains that produce SOD under the control of either a combined T7/lac promoter or the phi10 promoter were compared by using the following parameters: (a) total DNA content as an indicator of cell division, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellular SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of foreign protein synthesis also have the highest specific production rate. Cells, however, do not divide to a measureable degree but rather increase their size. The results confirm the importance of fine-tuning expression systems to prolong the lifetime of cells after induction. This will result in an increased yield.

Rational design of an improved induction scheme for recombinant Escherichia coli.

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.

Optimization of recombinant gene expression in Escherichia coli.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Amino-acid sequence comparison of nepovirus coat proteins.

The amino acid sequence of the coat proteins of several nepoviruses was determined by a combination of peptide and nucleic acid sequencing (grapevine fanleaf virus, arabis mosaic virus, tomato blackring virus, grapevine chrome mosaic virus). These sequences were compared and showed homologies ranging from 10% to 69%, and 96.7% for the two arabis mosaic virus strains. 10% homology does not reflect any relationship between viruses, and our results implicate, that nepoviruses, considering the homology of the coat protein sequences of viruses as a parameter for virus taxonomy, may be divided into several subgroups.

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus.

A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana.

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.

Detection of the trans activity of the plum pox virus NIa-like protease in infected plants.

The NIa-like protein of plum pox virus is a protease with high sequence specificity that is autocatalytically released from the viral polyprotein. In order to determine whether the protease is active in trans we constructed a fusion protein consisting of the C-terminal region of the plum pox virus polyprotein and the staphylococcal Protein A. The authentic protease recognition sequence Asn-Val-Val-Val-His-Gln-Ala occurs in the centre of this protein fusion. This protein was cleaved specifically by extracts of plum pox virus-infected plants due to the strong activity of the viral protease making it a useful tool for diagnostic purposes.

The complete nucleotide sequence of plum pox virus RNA.

The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.

Expression of the plum pox virus coat protein region in Escherichia coli.

A cDNA complementary to the 3' end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.

Early screening for anti-plum pox virus monoclonal antibodies with different epitope specificities by means of gold-labelled immunosorbent electron microscopy.

The technique of gold-labelled immunosorbent electron microscopy for the initial screening of monoclonal antibodies 10 days after cell fusion from 96-well culture plates is described. The technique is used to identify clones that secrete antibodies binding on the surface of the virion or to viral subunits, and compared to ELISA and Western blotting. High sensitivity was demonstrated.

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.