Role of environmental conditions on the expression levels,glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells

A recombinant CHO cell line in which the expresison of human follicle stimulating hormone (hFSH) was under the control of the actin promoter was maintained in steady state perfusion cultures on a protein free medium. The level of expression of the hFSH was controlled by varying the steady state level of dissolved oxygen (10–90% of air saturation) and of sodium butyrate (0–1.5mM). Under these conditions, the specific productivity of hFSH (q_FSH) varied from 0.7 to 4.8 ng hFSH/10⁶ cells/h. As the specific productivity of hFSH increased, there was a shift in the FSH isoforms to the lower pI fractions, corresponding to increased sialic acid content. As the specific productivity of hFSH increased, shifting the isoform distribution towards the lower pI isoforms, that the sialyltransferase enzymic activity also increased.     

A xyloglucan-oligosaccharide-specific α-d-xylosidase or exo-oligoxyloglucan-α-xylohydrolase from germinated nasturtium (Tropaeolum majus L.) seeds

The -xylosidase which is involved in the postgerminative mobilisation of xyloglucan in nasturtium seed cotyledons has now been purified to apparent homogeneity by a facile procedure involving lectin affinity chromatography. The purified enzyme, a glycoprotein, moved as a single band (apparent molecular weight 85000) on sodium dodecyl sulphate-gel electrophoresis, whilst isoelectric focusing gave a number of enzymatically active protein bands spanning the range pI = 5.0 to 7.1 (maximum activity at pI = 6.1). The enzyme did not hydrolyse the simple -xylosides p-nitrophenyl--d-xylopyranoside and woprimeverose (-d-Xyl(16)-d-Glc), or polymeric tamarind-seed xyloglucan. It released xylose from a complex mixture of oligosaccharides produced by exhaustive hydrolysis of tamarind seed xyloglucan using the xyloglucan-specific endo-(14)--d-glucanase from germinated nasturtium seeds (M. Edwards et al. 1986, J. Biol. Chem., 261. 9489–9494). The three xyloglucan oligosaccharides of lowest molecular size were purified from this mixture and were shown by ¹H-nuclear magnetic resonance (¹H-NMR) and enzymatic analysis to have the structures:Abbreviations Con A Concanavalin A - DEAE diethylaminoethyl - Gal galactose - Glc glucose - HPLC high-performance liquid chromatography - M _r apparent molecular mass - NMR nuclear magnetic resonance - pI isoelectric point - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Xyl xylose Much of the work reported in this paper was carried out with the aid of the European Community's Science Stimulation Action (Contract No. ST2P-0250-UK), and we wish to record our appreciation of this support.     

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for -hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to -hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of -hydroxy fatty acids which are formed in vitro.Abbreviations ABA abscisic acid - IEF isoelectric focusing - HHT hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase - pI isoelectric point     

Development of spectrophotometric method for monitoring trans-sialidase reaction and its application

A spectrophotometric method that can monitor the trans-sialidase catalyzed reaction was devised by using a chromogenic substrate, o-nitrophenyl--d-galactopyranoside (ONPG), as a sialic acid acceptor. This method yielded results that were consistent with those from HPLC, and could be very conveniently used in the screening of sialic acid donor or inhibitor of trans-sialidase.     

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of -fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a K _m for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.Abbreviations PEG polyethylene glycol - pI isoelectric point - PMSF phenylmethylsulphonyl fluoride We wish to acknowledge the support of the British/Swiss Joint Research Programme and the Sheffield University Research Support Fund. X.T. was in receipt of an Overseas Research Scholarship and a University of Sheffield Research Scholarship. We wish to thank Dr A. Moir for his help in N-terminal amino-acid sequencing.     

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H.Calcium and Cell Function III: 161–188, 1982). This active preparation consists of three related species containing residues 1–290, 1–296, and 1–298 of the 44.7 kD -subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740–11745, 1991). Good recoveries of catalytic activity — with varying degrees of calcium dependence — result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein.Physical characterization shows that the 33 kD chymotryptic fragment is globular, withs _20,w=2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylasea andb, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R.Biochem. Biophys. Res. Comm. 174: 344–350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[-P³²] ATP results in the reaction of 0.7 mol³²P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of³²P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser³⁰ while in the latter an additional reaction takes place at Ser⁸¹. The phosphopeptides correspond to sequences occurring in the -subunit of phosphorylase kinase.Abbreviations cAMP adenosine 3(,5(-cyclic monophosphate - NaDodSO₄ sodium dodecyl sulfate - Mops 3-(N-morpholino)propanesulfonic acid - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N(,N(-tetraacetic acid - ANS 1-anilinonapththalene-8-sulfonate - TPCK N-tosyl phenylalanyl chloromethyl ketone - TLCK N-tosyllysyl chloromethyl ketone - PTH phenylthiohydantoin The expected two to three fold increases in fluorescence intensity occur when assays are conducted with solutions containing 20 mM Tris chloride and no glycerophosphate.     

Primer dependent and independent forms of soluble starch synthetase from developing barley endosperms

The activity of soluble starch synthetase (ADP-glucose: -1,4-glucan -4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purified by ammonium sulfate precipitation and DEAE-cellulose chromatography and separated into four fractions. In the absence of an added carbohydrate primer two of the four fractions catalized the synthesis of a methanol-precipitable -glucan when high concentrations of sodium citrate and bovine serum albumim were added. The rate of -glucan synthesis by the unprimed reaction was higher than for the primed reaction. The four enzyme fractions were active with ADP-Glc, but not with UDP-Glc, both in the primed and in the unprimed reaction.Abbreviations ADP-Glc adenosine diphosphate glucose - DEAE-cellulose diethylaminoethyl-cellulose - DDT dithiothreitol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - kat katal (1 mol s^-1) - TRIS tris-(hydroxymethyl)-aminomethane - UDP-Glc uridine diphosphate glucose     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins inEscherichia coli

Summary The major leftward early promoter of phage p _L, has frequently been used to drive expression of heterologous genes inEscherichia coli.p _L is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressingp _L involves exposing cells to nalidixic acid, which results in the activation of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of theE. coli LexA protein, resulting in induction of the SOS regulon (at least 15E. coli genes, includingrec A). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind^–) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying acl⁺ defective lysogen. Synthesis of two heterologous proteins, human 1-antitrypsin and a fusion protein partially derived from thePlasmodium falciparum circumsporozooite surface antigen, was examined in the wild-type and mutant strains. The maximum -1 antitrypsin concentration achieved was improved by 50% when therecA o strain was used rather than the wild type; however; only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein wer observed. Use of thelexAind^– strain resulted in a decrease in the maximum concentration attained for both heterologous products.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Studies on the iodination of aras protein and the detection ofras polymers

Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greates incorporation of radioactivity with minimal modification of theras protein. Upon treatment of theras protein with [¹²⁵I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [¹²⁵I]ras was 6×10⁶ cpm/pmol totalras protein. Iondination did not alter the biological activity of theras protein as judged by its ability to bind GTPS and induced maturation ofXenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI ofras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. Theras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involvingras. Treatment of iodinatedras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purifiedras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.Abbreviations DSS Disuccinimidyl Suberate - GTPS Guanosine 5-[-thio] triphosphate - ATPS Adenosine 5[-thio] Triphosphate     

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA₁, GA₄ and GA₄-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA₈ and GA₃₄ did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA = 5-azido-7-[³H]indole-3-acetic acid - azido NPA = 5-azido-[3,6-³H]1-N-napthylpthalamic acid - BTP = 1,3-bis(Tris(hydroxymethyl)methylamino)-propane - GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate - [¹²⁵I]GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate - NPA = 1-Naphthylphthalmic acid - PAGE = Polyacrylamide gel electrophoresis - PMSF = phenylmethylsulfonyl fluoride - SDS = Sodium dodecyl sulphate - TLCK = L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization of-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^– phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^– wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.