Production, purification and characterization of recombinant human interferon gamma.

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.     

Theoretical studies on the mode of inhibition of ribonucleotide reductase by 2'-substituted substrate analogues

Several 2'-substituted-2'-deoxyribonucleotides are potent time-dependent inactivators of the enzyme ribonucleotide reductase (RNR), which function by destructing its essential tyrosil radical and/or by performing covalent addition to the enzyme. The former leads to inhibition of the R2 dimer of RNR and the latter to inhibition of the R1 dimer. Efforts to elucidate the mechanism of inhibition have been undertaken in the last decades, and a general mechanistic scheme has emerged. Accordingly, two alternative pathways lead either to the inhibition of R1 or R2, for which the 2'-chloro-2'-deoxynucleotides serve as the model for the inhibition of R1 and the 2'-azido-2'-deoxynucleotides the model for the inhibition of R2. However, the underlying reason for the different behavior of the inhibitors has remained unknown until now. Moreover, a fundamental mechanistic alternative has been proposed, based on results from biomimetic reactions, in which the 2'-substituents would be eliminated as radicals, and not as anions, as previously assumed. This would lead to further reactions not predicted by the existing mechanistic scheme. To gain a better understanding we have performed high-level theoretical calculations on the active site of RNR. Results from this work support the general Stubbe's paradigm, although some changes to that mechanism are necessary. In addition, a rational explanation of the factors that determine which of the dimers (R1 or R2) will be inactivated is provided for the first time. It has been demonstrated also that the 2'-substituents are indeed eliminated as anions, and not as radicals. Biomimetic experiments have led to different results because they lack a basic group capable of deprotonating the 3'-HO group of the substrate. It has been found here that the chemical character of the leaving group (radical or anionic) can be manipulated by controlling the protonation state of the 3'-HO group.     

Inhibition of folylpolyglutamate synthetase by substrate analogues with an ornithine side chain

N^α-[4-[[(4-Aminopteridin-6-yl)methyl]amino]benzoyl]-L-ornithine (dAPA-Orn) was synthesized, and its ability to inhibit folylpolyglutamate synthetase from mouse liver was compared with that of the corresponding 2,4-diamino analogue APA-Orn. Also compared were the inhibitory activities of the deaza analogues 5-deazaAPA-Orn, 8-deazaAPA-Orn, and 5,8-dideazaAPA-Orn, as well as those of N^α-pteroyl-L-ornithine (PteOrn) and its deaza analogues 5-deazaPteOrn and 5,8-dideazaPteOrn. The inhibition constant K_i of dAPA-Orn was 7-fold greater than that of APA-Orn, indicating that the 2-amino group plays a role in binding to the active site. The binding affinity of the 2,4-diamino compounds increased in the order 5-deazaAPA < APA-Orn <5,8-dideazaAPA-Orn < 8-deazaAPA-Orn, and that of the 2-amino-4(3H)-oxo compounds increased in the order 5-deazaPteOrn < PteOrn < 5,8-dideazaPteOrn. The most potent inhibitor of both groups was 8-deazaAPA-Orn, with a K_i of 0.018 μM, coresponding to an 8-fold and 15-fold increase in affinity relative to APA-Orn and 5-deazaAPA-Orn, respectively. The results suggest (a) that the binding of Orn-containing folylpolyglutamate synthetase inhibitors is affected to a greater degree by replacement of N⁸ by a carbon atom than it is by the corresponding change at N⁵, (b) that the effect of carbon for nitrogen replacement is greater in the 2,4-diamino derivatives than in the 2-amino-4(3H)-oxo compounds, and (c) that the 2,4-diamines are the better inhibitors. Comparison of the K_i values of the Orn-containing inhibitors with the K_m values of the corresponding glutamate-containing substrates revealed that K_m/K_i ratio can vary as much as 100-fold depending on the nature of the heterocyclic moiety, suggesting that caution should be exercised in using K_m values of known substrates to predict K_i values of putative inhibitors.     

3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in human cerebrospinal fluid. Storage and measurement by reversed-phase high-performance liquid chromatography and coulometric detection using 3-methoxy-4-hydroxyphenyllactic acid as an internal standard

To simultaneously measure 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) in human cerebrospinal fluid (CSF), we used an acetonitrile protein precipitation, reversed-phase high-performance liquid chromatography with coulometric detection, and 3-methoxy-4-hydroxyphenyllactic acid (MHPLA) as an internal standard for all three metabolites. MHPG, 5HIAA, HVA, and MHPLA were stable for one month when stored in CSF at -70 degrees C. Three determinations were made in triplicate for each of seven subjects over a 30-day storage period and the coefficients of variation within subject for these determinations ranged from 0.075 to 0.165 for MHPG, 0.045 to 0.148 for 5HIAA and 0.053 to 0.181 for HVA. Means and standard deviations of CSF concentrations were 10.7 +/- 3.0 ng/ml for MHPG, 22.4 +/- 9.9 ng/ml for 5HIAA, and 39.9 +/- 21.4 ng/ml for HVA. This method provides simple sample preparation, sensitivity, and cost advantages, as well as simultaneous extraction and quantitation of MHPG, 5HIAA, and HVA using an internal standard.     

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。     

Comparative conventional and microwave assisted synthesis of heterocyclic oxadiazole analogues having enzymatic inhibition potential

A comparative microwave assisted and conventional synthetic strategies were applied to synthesize heterocyclic 1,3,4-oxadiazole analogues as active anti-enzymatic agents. Green synthesis of compound 1 was achieved by stirring 4-methoxybenzenesulfonyl chloride ( a ) and ethyl piperidine-4-carboxylate ( b ). Compound 1 was converted into respective hydrazide ( 2 ) by hydrazine and then into 1,3,4-oxadiazole ( 3 ) by CS₂ on reflux. The electrophiles, N-alkyl/aralkyl/aryl-2-bromopropanamides ( 6a–p ) were synthesized and converted to N-alkyl/aralkyl/aryl-2-propanamide derivatives ( 7a–p ) by reaction with 3 under green chemistry. Microwave assisted method was found to be effective relative to conventional method. ¹³C-NMR, ¹H-NMR and IR techniques were availed to corroborate structures of synthesized compounds and then subjected to screening against lipoxygenase (LOX), α-glucosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. A number of compounds presented better potential against these enzymes. The most active compounds against LOX and α-glucosidase enzymes were subjected to molecular docking study to explore their interactions with the active sites of the enzymes.     

Chemical extraction and direct adsorptive purification of recombinant human antigen Ro52

The antigenic protein Ro52 was expressed in the E. coli system harboring a 6 x His tag in the form of insoluble inclusion bodies. Direct chemical extraction of the product using 6-8 M urea proved to be effective. Furthermore, the tagged protein was recovered by direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiacetic acid. Screening experiments in small packed columns revealed that selective binding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamic evaluation of scaled-up fluidized systems showed values for the phi (dynamic zone) parameter in the range 0.95-1.00 for fluidization in buffer and in the range 0.70-0.85 for the biomass-containing feedstock. Removal of macromolecular DNA released by the disrupted biomass was mandatory. Under optimized process conditions good recovery (60-70%) was achieved and a highly purified (95%) product obtained. The purified Ro52 retained its immunoreactivity against sera of patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome-related disorders. The production and application of new recombinant antigens may contribute to increasing the sensitivity and specificity of the detection of anti-Ro antibodies in these autoimmune diseases.     

Purification of human 6-phosphogluconate dehydrogenase from human haemolysate with chromatography on an immobilized dye as the essential step and use of automation. Simultaneous purification of lactate dehydrogenase

The screening procedure described in the preceding paper allowed a practical purification procedure to be devised that was automated for human 6-phosphogluconate dehydrogenase. The purification needed only two chromatographic steps, first on immobilized Procion Blue HE-GN and then on Phenyl-Sepharose. This technique also gave purified lactate dehydrogenase. Both enzymes showed single bands in SDS polyacrylamide gel electrophoresis.     

Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8?μU/ml. Recoveries ranged between 33.2?% and 51.7?%. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301?μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog?, Novolog?, Apidra?, Lantus?, and Levemir? in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.     

One‐step refolding and purification of recombinant human tumor necrosis factor‐α (rhTNF‐α) using ion‐exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic‐based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor‐α (rhTNF‐α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF‐α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF‐α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10⁸ IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF‐α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF‐α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.     

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.     

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.     

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting‐out

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine‐tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni‐chelated Sepharose Fast‐Flow affinity column, ammonium sulfate salting‐out and Sephadex G‐75 size‐exclusion column in turn. The purity analysis by SDS–PAGE, high‐performance size‐exclusion and reversed‐phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti‐angiogenic activity under the effective range of 5.0–25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.