Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli

A physiologically relevant thrombopoietin (TPO) must be a humoral regulator with lineage specificity for megakaryocytes and their precursors. It should be capable of stimulating platelet production in normal animals, and elevated levels of TPO should be detectable in the plasma following acute, severe thrombocytopenia. Acute thrombocytopenia provides a model system that is likely to predict the effects of TPO, since many of the effects on megakaryocytes and platelets observed after induction of acute thrombocytopenia would be mediated by TPO. Important questions remain to be answered. Do the currently available data for the c-Mpl ligand explain previously published data that describe elevated levels of Meg-CSF in the circulation following production of bone marrow aplasia? Does the c-Mpl ligand account for all of the megakaryocyte stimulatory factors that have been described? Is there another factor that accounts for at least some of the acute alterations in megakaryocytopoiesis that occur immediately following a decrease in platelet levels?     

On the kinetics of phase separation in aqueous two-phase systems

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R. relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.     

Predictable deuteration of recombinant proteins expressed in Escherichia coli

Carrageenan was used to study inflammation-induced changes in spinal nociception and its brain stem modulation in the pentobarbitone-anesthetized rat. Carrageenan was administered intraplantarly into one hindpaw 2 h before the start of electrophysiological single unit recordings of wide-dynamic range (WDR) neurons of the spinal dorsal horn. Carrageenan produced a significant leftward shift in the stimulus-response function for mechanical stimuli, whereas that for noxious heat stimuli was short of statistical significance. Conditioning electrical stimulation in the rostroventromedial medulla (RVM) significantly attenuated noxious heat-evoked, but not mechanically evoked, responses to spinal dorsal horn WDR neurons in the control (contralateral) side. However, in the carrageenan-treated side RVM stimulation had no significant effect on mechanically or noxious heat-evoked responses. Following direct spinal administration of neuropeptide FF (NPFF), noxious heat-evoked responses, but not mechanically evoked responses, were attenuated by RVM-stimulation also in the carrageenan-treated side. This selective NPFF-induced enhancement of brain stem-spinal inhibition was not reversed by naloxone. The results indicate that carrageenan-induced inflammation significantly changes the response properties of spinal nociceptive neurons and their brain stem-spinal modulation. During inflammation, NPFF in the spinal cord produces a submodality-selective potentiation of the antinociceptive effect induced by brain stem-spinal pathways, independent of naloxone-sensitive opioid receptors.     

Purification and characterization of D-beta-hydroxybutyrate dehydrogenase expressed in Escherichia coli

D-beta-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 mumol/(min.mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.     

Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.     

水-正丙醇-硫酸铵体系析相萃取分离钯的研究

研究了水-正丙醇析相萃取Pd(Ⅱ)的行为及Pd(Ⅱ)与一些金属离子分离的条件。结果表明, 硫酸铵能使正丙醇的水溶液分成两相,在分相过程中,Pd(Ⅱ)与溴化钾生成的PdBr₄^2-与质子化正丙醇 (C₃H₇OH₂⁺)形成缔合物PdBr₄^2-[C₃H₇OH₂⁺]₂能被正丙醇相完全萃取。当溶液中正丙醇的体积分数、溴化钾浓度和硫酸铵的质量浓度分别为30 %, 7.0×10-3 mol/L, 0.2 g/ mL且pH 2.0时, Pd的萃取率达到96.7%以上,而Ag(Ⅰ)、Cu(Ⅱ)、Fe(Ⅲ)、V(Ⅴ)、Ni(Ⅱ)、W(Ⅵ)、Al(Ⅲ)、Pb(Ⅱ)、Cr(Ⅲ)、U(Ⅵ)、Mg(Ⅱ)、Bi(Ⅲ)基本不被萃取,实现了Pd(Ⅱ)与上述金属离子的分离。对合成水样和钯镍电镀液中的钯进行了定量萃取分离和测定,获得了满意结果。     

Purification and crystallization of the CDK-associated protein phosphatase KAP expressed in Escherichia coli

The kinase associated phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates the cell cycle control protein, cyclin dependent kinase-2 on Thr 160 in a cyclin dependent manner (Poon & Hunter, 1995). We report here the over-expression of KAP in Escherichia coli as an N-terminal His-tagged protein using a modified pET-28a T7-expression vector. The recombinant protein was purified to homogeneity and crystallized. The crystals diffract to 2.3 A resolution when exposed to synchrotron radiation and belong to space group P6(1)22, or its enantiomorph P6(5)22, with unit cell dimensions a = b = 74.5 A, c = 139.5 A.     

Purification and characterization of Streptococcus mutans glucosyltransferase (GtfC) expressed in Escherichia coli

Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.     

Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; beta-D-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10 degrees C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg-1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.     

Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.     

Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes

We expressed two regions of the serine repeat antigen (SERA) protein of Plasmodium falciparum in Escherichia coli by synthesizing the genes with a changed codon usage. One of the synthetic gene sequences encodes amino acid residues 17-382 (SE47') and the other encodes amino acid residues 586-802 (SE50A). The products produced by the synthetic gene sequences in E. coli accounted for 15-30% of the total bacterial protein. Antisera against both the purified gene products prepared in rats inhibited malaria parasite growth in vitro. The anti-SE47' serum was significantly more inhibitory than the anti-SE50A serum. The described methods provide a large scale preparation of recombinant antigens for improving and producing malaria vaccine.     

Characterization of human immunodeficiency virus type 1 integrase mutants expressed in Escherichia coli

The eight mutant integrase (IN) proteins of human immunodeficiency virus type (HIV-1), which have a single point mutation at a highly conserved central region, were prepared, and characterized in terms of their endonucleolytic activities and disintegration activities in vitro. Mutation of two highly conserved amino acids, Asp116 or Glu152, leads to complete loss of both the activities, suggesting that these two amino acids are directly associated with enzymatic functions. In addition, the mutant of the position Ser147 was found to have highly depressed endonucleolytic activity showing that the reaction was very delayed in comparison with that of the wild type. However, significant disintegration was detected in the mutant Ser147, indicating that the enzymatic mechanisms of the endonucleolytic and disintegration activities are not exactly reverse. The integrase protein with a mutation at the conserved amino acid Asn117 or Gly118 had a slight loss of the endonucleolytic activity, while a mutation at the three positions, Tyr143, Ser153, and Lys159, had no detectable effect on their enzymatic activities. These results indicate that only a few of the conserved amino acids are critical for enzymatic activities.     

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K(m) = 0.70 microM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.     

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.     

Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis

A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized. Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism. Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene. The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate. The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions. Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site. The divalent cations Mg2+, Zn2+ and Mn2+ were found to be inhibitors of enzymatic activity, whereas Ca2+ had no effect. Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity. A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.     

Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in Escherichia coli

Hyaluronan is a major component of synovial fluid. It is synthesized in the joint and partly degraded in joint capsule and partly carried by lymph to lymph nodes and the general circulation. During various joint disorders, especially inflammatory ones, the amount of hyaluronan in the joint and its turnover rate are increased leading to pathologically high serum levels of the polysaccharide. These levels are a complex function of synovitis mass and the physical activity of the patient. Intra-articular injection of hyaluronan has been used in the treatment of osteoarthritis. Although it results in a relief of pain, controlled clinical trials have not yet convincingly shown any beneficiary effects of the treatment.     

Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli

Bacteriorhodopsin (BR) from Halobacterium salinarum as well as halorhodopsin (pHR) and sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were functionally expressed in E. coli using the method of Shimono et al. IFEBS Lett. (1997) 420, 54-56]. The histidine tagged proteins were purified with yields up to 1.0 mg/l cell culture and characterized by ESI mass spectrometry and their photocycle. The pSRII and pHR photocycles were indistinguishable from the wild type proteins. The BR photocycle was considerably prolonged. pSOII is located in the cytoplasmic membrane and the C-terminus is oriented towards the cytoplasm as determined by immunogold labelling.     

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.     

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.     

氯化钠-硫氰酸钾-正丙醇体系析相萃取分离和富集钛

研究了正丙醇-硫氰酸钾-水体系析相萃取分离和富集Ti的行为及与一些金属离子分离的条件。结果表明, 氯化钠能使正丙醇的水溶液分成两相,在分相过程中, Ti和硫氢化钾生成的Ti(SCN)₆^2-与质子化正丙醇C₃H₇OH₂⁺ 形成的缔合物［Ti(SCN)₆^2-］［C₃H₇OH₂⁺］₂能被正丙醇相完全萃取。当正丙醇、硫氰酸钾和氯化钠的浓度分别为30%（V/V)、8.0×10^-2 mol/L和0.17 g/mL时, Ti的萃取率达到98.9%以上,V、Ce、Mg、Mn、Cd、Cr、Al、Fe、Zn、Ni、Ag 和 W不被萃取,实现了Ti与上述金属离子的分离。对合成水样中的钛进行分离和测定, 结果满意。