Yanling Xie

Effects of Modification of Amino Groups with Poly (Ethylene Glycol) on a Recombinant Uricase from Bacillus fastidiosus

After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).

Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions

Bacillus fastidious uricase (BF uricase) containing 322 amino acid residues exhibited high stability under physiological conditions. Its crystal structure was solved to 1.4-Å resolution, showing homotetramer containing two homodimers. After the intersubunit antiparallel β-sheet in its homodimer, each subunit had a total of 18 C-terminus residues forming an α-helix (Q305-A313) and random coil (S314-L322) on surface to bury other two α-helices (I227-T238 and I244-R258). In comparison, reported crystal structures of Arthrobacter globiformis and Aspergillus flavus uricases had atomic coordinates of only some C-terminus residues, while the crystal structures of all the other uricases accessible before September 2014 missed atomic coordinates of all their C-terminus residues, after the intersubunit antiparallel β-sheets. In each homodimer of BF uricase, H-bonds were found between E311 and Y249 and between Y319 and D257; electrostatic interaction networks were found to surround D307 plus R310 and intersubunit R3, K312 plus D257, E318 plus K242, and L322 plus R258. Amino acid mutations that disrupted those interactions when R3 and D307 were reserved caused moderate decreases of activity at pH 9.2 while negligible decreases of activity at pH 7.4, but destroyed stability at pH 7.4 while slightly decreased stability at pH 9.2. Such structural information guided the fusion of 6His-tag to the C-terminus of the mutant L322D with SNSNSN as a linker to reserve the activity and stability. Hence, the folding of the C-terminus residues is crucial for thermal stability of BF uricase under physiological conditions; these new structural insights are valuable for molecular engineering of uricases.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λ(A)) much larger than that of Product B (λ(B)) formed by Enzyme B action on Substrate B; λ(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ(0)); (b) absorbance at λ(A) and λ(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases p-nitroaniline from γ-glutamyl-p-nitroaniline with λ(0) at 344 nm and λ(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λ(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and β-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ(0) at 405 nm and λ(A) at 458 nm, β-D-galactosidase releases 4-nitrophenol from β-D-(4-nitrophenyl)-galactoside with λ(B) at 405 nm. No interference from substrates/products made SDESA of β-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

BackgroundFor screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.ResultsDuring uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity.ConclusionSpecific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Reversible Inactivation of an Intracellular Uricase from Bacillus fastidiosus via Dissociation of Homotetramer into Homodimers in Solutions of Low Ionic Strength

An intracellular uricase from Bacillus fastidiosus with high catalytic capacity and strong resistance to xanthine was inactivated in water but could be essentially re-activated in solutions of high ionic strength. By polyacrylamide gel electrophoresis (PAGE), gradient PAGE, sodium-dodecyl-sulfate-PAGE, gel-filtration through Sephadex G200, and activity staining with peroxidase and its chromatogenic substrate, this homotetrameric uricase in water was found to dissociate into inactive homodimers that could form active homotetramers again in solutions of high ionic strength. Sensitivity to low ionic strength of solutions complicates formulation of this uricase as a drug and its elimination requires protein engineering.

A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia.

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.

Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.

By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

Soluble Expression in Escherichia coli of Active Human Cyclic Nucleotide Phosphodiesterase Isoform 4B2 in Fusion with Maltose-Binding Protein

Recombinant expression in Escherichia coli of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4B2) fused to maltose-binding-protein (MBP-hPDE4B2) was investigated. hPDE4B2 DNA amplified via nested RT-PCR with total RNAs from U937 cells was ligated with pMAL-p2x. After induction at 18 °C for 16 h, soluble MBP-hPDE4B2 was produced in E. coli. MBP-hPDE4B2 after amylose-resin chromatography showed 35% homogeneity, and its Michaelis-Menten constant was 10±2 μM (n=3). Rolipram had a dissociation constant of 9±2 nM (n=2), and zinc ion was a potent inhibitor. Hence, MBP-hPDE4B2 was expressed in E. coli as a soluble active protein.

Fluorometric Titration Approach for Calibration of Quantity of Binding Site of Purified Monoclonal Antibody Recognizing Epitope/Hapten Nonfluorescent at 340 nm

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

Method to screen aromatic ligands in mixtures for quantitative affinities to target using magnetic separation of bound ligands along with HPLC and UV photometry detection

AbstractA new screening method was tested to estimate the affinity of an aromatic ligand in a mixture through competitive binding against an exogenous reference ligand. The target protein was immobilized on magnetic particles and one or several ligand(s) in each reaction mixture were prepared by parallel combinatorial-synthesis without purification. Specifically, the binding of aromatic biotin derivatives to streptavidin immobilized on magnetic particles was used as the model. An approximation equation that works under the condition of binding ratios below 0.1 for the candidate ligand and the reference ligand was developed. It was found that the relative affinity of each biotinylated aromatic candidate ligand in mixtures was consistent with that by using HPLC-MS analyses or by a homogenous method using its purified counterpart. Hence, this new screening method using HPLC-UV is considered to be highly effective. FigureThe representative procedure to realize the new screening technique for quantitative affinity of an aromatic candidate ligand of unknown quantity in a mixture sample