The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1 = isoform 2 < isoform 4 < isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.     

Refolding,purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca²⁺ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

Expression of recombinant human cathepsin K is enhanced by codon optimization

A synthetic codon-optimized gene encoding human procathepsin K has been cloned in Escherichia coli using pET28a+ vector. The recombinant His-tagged fusion protein was expressed as inclusion body, solubilized in urea and purified by metal affinity chromatography. The purified protein was refolded by dilution technique, concentrated and finally purified by gel-filtration chromatography. The expressed protein was confirmed by Western blot analysis with human cathepsin K specific antibody. We have obtained 140 mg purified and refolded protein from 1 L bacterial culture which is the highest (nearly three times higher) yield reported so far for a recombinant human procathepsin K. The protease could be autocatalytically activated to mature protease at lower pH in presence of cysteine protease specific activators. The recombinant protease showed gelatinolytic and collagenolytic activities as well as activity against synthetic substrate Z-FR-AMC with a K_m value of 5 ± 2.7 μM and the proteolytic activity of the enzyme could be blocked by cysteine protease inhibitors E-64, leupeptin and MMTS.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

Crucial factors affecting the nitrile hydratase production of Mesorhizobium sp. F28

Mesorhizobium sp. F28 contains cobalt-NHase, which effectively converts acrylonitrile into acrylamide. When urea was added to the culture medium, the NHase activity was 62.3 U ml^?1 (R2A–R2A/urea) after 22.5 h of cultivation, which was similar to that in the medium without addition (R2A–R2A, 70.0 U ml^?1). The relative activity of the purified NHase was 100%, 92%, 94%, and 92% in the medium containing, respectively, 0 mM, 2 mM, 5 mM, and 10 mM of urea. Urea had no significant effect on the purified NHase activity of Mesorhizobium sp. F28. This research did not observe the NHase production by Mesorhizobium sp. F28 when acrylonitrile was supplemented in the culture medium except that cobalt ions existed. The highest enzyme activity was 328.5 U ml^?1 as cobalt ions were added in the pre-culture and culture medium after 22.5 h of cultivation (R2A/Co-R2A/Co); compared to media without cobalt ions (R2A–R2A, 22.5 h, 70.5 U ml^?1) this is an almost five-fold enhancement. It can be concluded that culture media containing cobalt ions was beneficial for the formation of active NHase of Mesorhizobium sp. F28.     

Solution conditions define morphological homogeneity of α-synuclein fibrils

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10 mM Tris–HCl, 10 mM NaCl and 0.1 mM EDTA at pH 7.4 display a narrow distribution of periodicities with an average value of 102 ± 6 nm for the A140C mutant and 107 ± 9 nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Characterization of variant diphtheria toxin–interleukin-3 fusion protein,DTIL3K116W,for phase I clinical trials

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin–interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at ?80 °C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography–HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296 EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC₅₀ of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC₅₀ of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at ?80 °C. Further, the drug was stable at 4 and 25 °C in the plastic syringe and administration tubing for 48 h.     

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K_m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.     

Regeneration of denatured polyphenol oxidase in Toona sinensis (A.Juss.) Roam

The regeneration of polyphenol oxidase (PPO) from leaves of Toona sinensis (A.Juss.) Roam (TS), denatured by boiling with 0.4% (w/v) sodium dodecyl sulfate (SDS) and 1% (v/v) β-mercaptoethanol, and was studied with SDS-polyacrylamide gel electrophoresis (PAGE) and the gel-activity-stained assay. The optimal regenerating conditions for the denatured TS-PPO were as follows: extracting with buffer of 150 mM Na-Pi, pH 7.2; regenerating with buffer of 50 mM Tris–HCl, pH 7.2, 2% Triton X-100 for 45 min. In addition, the PPO regeneration was promoted by the addition of Cu²⁺ in the recovery buffer. The regenerating activity of the denatured-PPO was reduced in samples extracted with buffers containing polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) or Triton X-100.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Simple liquid chromatography method for the quantification of irinotecan and SN38 in sheep plasma: Application to in vivo pharmacokinetics after pulmonary artery chemoembolization using drug eluting beads

A rapid and simple liquid chromatography–fluorescence detection (LC–FD) method was developed and validated for the simultaneous quantification of irinotecan (CPT11) and SN38 in sheep plasma. Camptothecin (CPT) was used as the internal standard. A single step protein precipitation with acetonitrile was used for sample preparation. The separation was achieved using a 5 μm C18 column (250 mm × 4.5 mm, 5 μm) with a mobile phase composed of 36 mM sodium dihydrogen phosphate dehydrate and 4 mM sodium 1 heptane sulfonate–acetonitrile (72:28), the pH of the mobile phase was adjusted to 3. The flow rate was 1.45 mL/min and the fluorescence detection was operated at 355/515 nm (excitation/emission wavelengths). The run time was 13 min. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 5 ng/mL for both CPT11 and SN38. The assay was linear over concentrations ranging from 5 to 5000 ng/mL and to 240 ng/mL for CPT11 and SN38, respectively. This method was used successfully to perform plasma pharmacokinetic studies of CPT11 after pulmonary artery embolization (PACE) in a sheep model. It was also validated for CPT11 and SN38 analysis in sheep lymph and human plasma.     

Solvent free biocatalytic synthesis of isoniazid from isonicotinamide using whole cell of Bacillus smithii strain IITR6b2

A biocatalytic route for the synthesis of isoniazid, an important first-line antitubercular drug, in aqueous system is presented. The reported bioprocess is a greener method, does not involve any hazardous reagent and takes place under mild reaction conditions. Whole cell amidase of Bacillus smithii strain IITR6b2 having acyltransferase activity was utilized for its ability to transfer acyl group of isonicotinamide to hydrazine–2HCl in aqueous medium. B. smithii strain IITR6b2 possessed 3 folds higher acyltransferase activity as compared to amide hydrolase activity and this ratio was further improved to 4.5 by optimizing concentration of co-substrate hydrazine–2HCl. Various key parameters were optimized and under the optimum reaction conditions of pH (7, phosphate buffer 100 mM), temperature (30 °C), substrate/co-substrate concentration (100/1000 mM) and resting cells concentration (2.0 mg_dcw/ml), 90.4% conversion of isonicotinamide to isoniazid was achieved in 60 min. Under these conditions, a fed batch process for production of isoniazid was developed and resulted in the accumulation of 439 mM of isoniazid with 87.8% molar conversion yield and productivity of 6.0 g/h/g_dcw. These results demonstrated that enzymatic synthesis of isoniazid using whole cells of B. smithii strain IITR6b2 might present an efficient alternative route to the chemical synthesis procedures without the involvement of organic solvent.     

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl₂, 30 mM KCl, 2.0 mM (NH₄)₂SO₄, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low μM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH₂, exhibited a K_d for hMC4R of 9.1 ± 1.4 μM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Characterization and flocculating properties of an extracellular biopolymer produced from a Bacillus subtilis DYU1 isolate

Biopolymer DYU500 produced from Bacillus subtilis DYU1 was found to have excellent flocculating ability. With the addition of 40 mg-DYU500/L and 50 mM CaCO₃, the optimum temperature for flocculation performance of DYU500 was 30 °C, giving the highest flocculating activity and rate of 13.5 and 97%, respectively. Analysis with Fourier transform infrared spectrophotometry (FT-IR), nuclear magnetic resonance spectrometry (NMR) and amino acid identification shows that the DYU500 biopolymer mainly possesses the structure of poly-glutamic acid (PGA). The average molecular weight of DYU500 was about (3.16–3.20) × 10⁶ Da as determined by gel permeation chromatography. The major components of biopolymer DYU500 were total sugars, uronic acids, proteins and polyamides (homopolymer of glutamic acid), accounting for a weight ratio of approximate 14.9, 2.7, 4.4 and 48.7% (w/w), respectively. The flocculating activity of DYU500 in the kaolin suspension was markedly stimulated by the addition of bivalent cations Ca²⁺ or Mg²⁺ in optimum concentration ranges of about 0.15–0.90 and 0.10–0.90 mM, respectively. The synergistic effect of cations was most effective at a weak acidic or neutral pH (6.0–7.0). The flocculating activity of DYU500 linearly decreased with an increase in incubation temperature and the activity was completely lost when heating upon 120 °C, arising from the destruction of the polyamides structure of DYU500. Moreover, mechanisms describing the flocculation process with DYU500 were proposed based on the experimental observations.