Numerous human infections with avian influenza viruses in Asia in recent years have raised the concern that the next influenza pandemic is imminent. The most effective way to combat influenza is ...through the vaccination of the public. However, a minimum of 3–6 months is needed to develop an influenza vaccine using the traditional egg-based vaccine approach. The influenza hemagglutinin protein (HA), the active ingredient in the current vaccine, can be expressed in insect cells using the baculovirus expression vector system and purified rapidly. An influenza vaccine based on such a recombinant antigen allows a more timely response to a potential influenza pandemic. Here, we report an innovative monitoring assay for recombinant HA (rHA) expression and a rapid purification process. Various biochemical analyses indicate that the purified rHA is properly folded and biologically active.
Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of
Escherichia coli alkaline phosphatase in which two of the three active-site metal ions ...have a direct role in catalysis. Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 Å to final working
R-factors of 15.4 % and 16.4 %, respectively. In the refinement of both structures, residues in the active sites were treated anisotropically. The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product. In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile. These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported.
The proposed double in-line displacement mechanism of
Escherichia coli alkaline phosphatase (AP) involving two-metal ion catalysis is based on NMR spectroscopic and X-ray crystallographic studies. ...This mechanism is further supported by the X-ray crystal structures of the covalent phospho-enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild-type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site’s metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.
The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a ...purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (~50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.
Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in ...the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form.
The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach.
rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 Ã resolution. The vanadate ion takes on a trigonal
bipyramidal ...geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies
the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized
by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line
displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition
state in the enzyme-catalyzed reaction.
One of the best‐studied examples of a class A β‐lactamase is Escherichia coli TEM‐1 β‐lactamase. In this class of enzymes, the active‐site serine residue takes on the role of a nucleophile and ...carries out β‐lactam hydrolysis. Here, the structures of the wild‐type and the S70G enzyme determined to 1.55 and 2.1 Å, respectively, are presented. In contrast to the previously reported 1.8 Å structure, the active site of the wild‐type enzyme (1.55 Å) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X‐ray crystal structure of the S70G mutant has an altered Ser130 side‐chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild‐type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active‐site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.
NADH:cytochrome b(5) reductase (FpD) is an enzyme capable of converting the prodrug mitomycin C (MC) into a DNA alkylating agent via reduction of its quininone moiety. In this study, Chinese hamster ...ovary (CHO) cells were transfected with a cDNA encoding rat FpD. Despite the demonstrated ability of this enzyme to reduce MC in vitro, a modest 5-fold level of overexpression of FpD activity in CHO cells did not increase the cytotoxicity of the drug over that seen with the parental cell line under either aerobic or hypoxic conditions. When the enzyme, which is predominantly localized in the mitochondria, was instead directed to the nucleus of cells by the fusion of the SV40 large T antigen nuclear localization signal sequence to the amino terminus of an FpD gene that lacked the membrane anchor domain, drug sensitivity was significantly enhanced at all concentrations of MC examined (2-10 microm) under both aerobic and hypoxic conditions, with greater cell kill occurring under hypoxia. The marked increase in drug sensitivity under hypoxia at 10 microm MC corresponded to a measurable increase in total MC-DNA adducts at the same concentration. The results indicate that the cytotoxicity of MC is modulated by the subcellular location of FpD, with greater cell kill occurring when bioactivation occurs in the proximity of its target, nuclear DNA.
It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 102-fold slower than ...phosphate monoesters. This “thio effect” is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this ∼23-fold change in the magnitude of the thio effects, the magnitudes of Brønsted βlg for the native AP (−0.77 ± 0.09) and the R166A mutant (−0.78 ± 0.06) are the same. The identical values for the βlg indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the βlg indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.
Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D
+ myelomonocytic leukemia cells interferes with their capacity to respond to all-
trans retinoic acid (ATRA) induced ...differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.
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