Isolation of 3,4-dihydroxyphenylalanine-containing proteins using boronate affinity chromatography

A rapid procedure for the isolation of 3,4-dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.0 M ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5.     

Crystallization and preliminary diffraction studies of NodL, a rhizobial O-acetyl-transferase involved in the host-specific nodulation of legume roots.

The NodL specified O-acetyltransferase from the microbial symbiont Rhizobium leguminosarum has been over-expressed in Escherichia coli and purified using affinity-elution dye chromatography as the key step. The protein has been crystallized at 20 degrees C in 18% PEG 600, 0.1 M Tris/HCl buffer, pH 8.5, containing 1% dioxane, 0.25% octyl-beta-glucoside, and 5 mM coenzyme A using the hanging drop vapor diffusion method. Ambient temperature X-ray diffraction studies reveal the space group to be hexagonal (P6(3)22) with lattice constants a = b = 77.08 A, c = 160.6 A, and alpha = beta = 90 degrees, gamma = 120 degrees. Crystals that are flash-frozen to 120 K diffract beyond 2.7 A.     

Purification of human leukocyte elastase and cathepsin G by chromatography on immobilized elastin

Human leukocyte elastase and cathepsin G were isolated from purulent sputum by a simple procedure involving chromatography on elastin-agarose. Salt extracts of sputum were prepared, treated with DNase, and the precipitate which formed extracted and applied to a column of soluble elastin-Sepharose 4B. Contaminating protein was eluted with 50 mM Tris, 50 mM NaCl, pH 8.0 and then two column volumes of 50 mM acetate, 1.0 M NaCl, pH 5.0. The tightly bound elastase and cathepsin G together with a trypsin-like serine protease could finally be eluted with 50 mM acetate, 1.0 M NaCl, 20% DMSO, pH 5.0. Resolution of the proteases was accomplished by cation-exchange chromatography. Disc gel electrophoresis established the purity of elastase and cathepsin G and confirmed the existence of several isozymes for each.     

Intestinal surface peptide hydrolases: identification and characterization of three enzymes from rat brush border.

Peptide hydrolases were solubilized from rat small intestinal brush border by papain and separated by Sephadex G-200 chromatography, velocity gradient ultracentrifugation and polyacrylamide disc electrophoresis and designated according to approximate molecular size from sedimentation studies. Peptidases I (apparent Mr 230 000) and II (apparent Mr 160 000) are oligopeptidases with maximum specificity for tripeptides with identical pH optima (7.5) and similar apparent Km with L-Leu-Gly (I, 0.60 MM; II, 0.76 mM). L-Leucyl-beta-naphthylamide is a competitive inhibitor of both enzymes. Concentration of peptidase II produced partial conversion to peptidase I on polyacrylamide disc electrophoresis. The third peptide hydrolase (III, Mr 120 000) is a dipeptidase with pH optimum 8.5 and apparent Km for L-Leu-Gly of 0.65 mM. These peptide hydrolases were inhibited appreciably (37-59%) by 0.2 M glycine/NaOH, Tris - HCl or Tris - glycine buffers. EDTA (5 mM) completely inhibited these enzymes but all activity was restored by dialysis against buffer without divalent ions. Subsequent addition of Mg2+, Mn2+, Co2+ or Zn2+ (1-2 mM) inhibited peptidases I and II variably (4-81%) depending upon the substrate and buffer used. In contrast peptidase III was activated slightly by metal ions (5-20%). These peptide hydrolases are strategically located at the intestinal lumen-cell interface and possess biochemical characteristics making them ideally suited to play a pivotal role in the final stage of protein digestion.     

Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: decrease in the stability by electrodialysis and recovery by addition of the magnesium

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non-electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon-like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non-electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.     

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.     

Adsorption of human serum proteins onto TREN-agarose: Purification of human IgG by negative chromatography

Tris(2-aminoethyl)amine (TREN) – a chelating agent used in IMAC – immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90–95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: study of long-range stability

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The stability of this hexagonal lattice structure in long-term incubation at 4 C was investigated. The hexagonal lattice structure was stable for at least 220 days when the LPS was suspended in distilled water, but it had been disintegrated into a rough mesh-like structure when the LPS was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.5, at 4 C for 60 days. Half of the Mg bound to the LPS was released when the LPS was suspended in Tris buffer for 60 days, whereas Mg was not released when it was suspended in distilled water even for 220 days. By contrast, it was stable for at least 220 days in Tris buffer containing 5 mM MgCl2. The LPS suspended in Tris buffer for 60 days, at which time the structure had been disintegrated, could be restored to the original hexagonal lattice structure within 24 hr by addition of 5 mM MgCl2. From these results it is concluded that the hexagonal lattice structure of the LPS retains long-range stability if Mg bound to the LPS is not released from the LPS.     

A rapid method for the assay of guanylate cyclase.

An extremely rapid and sensitive assay for guanylate cyclase utilizing [alpha-32P]-GTP has been developed. It involves incubation of 5-100 mug of enzyme protein with 1 mM [alpha-32P]-GTP in 40 mM Tris HC1 buffer (pH 7.4) containing 3-3 mM MnSO2, 10 mM theophylline and 1 mM cyclic GMP. The reaction is terminated by addition of EDTA, and [32P]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H+ and alumina. Recovery of 75-85% of [3H]-cyclic GMP and a blank of 0.001-0.003% of added [32P]-GTP was routinely obtained. The [32P] radioactivity isolated was shown to be cyclic GMP by a variety of techniques. The assay has also been shown to be applicable for a variety of tissues.     

Isolation of human interferon β1b (Ser17) expressed in <Emphasis Type="Italic">E. coli</Emphasis> with the use of ion-exchange chromatography

A method for isolation of interferon β1b (Ser17) from the inclusion bodies is presented. This method consists of the following stages: solution and reduction of the protein from the inclusion bodies, refolding, chromatography on DEAE-sepharose, chromatography on SP-sepharose, concentration of the protein solution, demineralization, and addition of stabilizers. The refolding was performed by dilution of the solution of the reduced protein with a buffer (pH 8.0) containing 50 mM Tris-HCl, 25 μM CuCl₂, and 0.5% Tween-20. Interferon β1b was eluted from a cation-exchange column with a pH gradient (9.3–11.3) of sodium phosphate buffer. The eluate was concentrated and desalted on a column with sephadex G-50 in 1 mM NaOH, mannitol, and sodium phosphate were added for neutralization of the protein solution. The product was homogenous according to gel electrophoresis and HPLC, and exhibited the practically same antiproliferative activity as that of Betaferon taken as a standard. Thus, a possibility of preparation of the purified and active interferon β by ion-exchange chromatography in the presence of the Tween-20 nonionic detergent was demonstrated. The proposed procedure is promising for application to large-scale production and industry.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

Discrete soluble forms of middle and high molecular weight neurofilament proteins in dilute aqueous buffers

Neurofilament proteins purified from bovine spinal cord were characterized by sedimentation studies in aqueous buffers. In 10 mM Tris, pH 8, the middle molecular weight neurofilament protein (NF-M) has a sedimentation coefficient, S20,w, of 2.6 S. Sedimentation equilibrium data shows considerable nonideality; extrapolation to infinite dilution and correction for the primary charge effect yield a molecular weight of 1.09 X 10(5), indicative of a monomeric structure. When the ionic strength was increased, the sedimentation coefficient increased slightly, and the protein began to form larger aggregates. Reconstitution of short intermediate filaments was observed upon dialysis of denatured NF-M versus a reconstitution buffer. A circular dichroism spectrum of NF-M in 10 mM Tris was typical of alpha + beta proteins. High molecular weight neurofilament protein (NF-H) showed a considerable tendency to aggregate in 10 mM Tris, but a principal species with a sedimentation coefficient of 3.2 S was observed, and sedimentation equilibrium data also suggest a monomeric structure.