Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.     

Negative purification method for the selection of specific antibodies from polyclonal antisera

We developed a protocol to remove non-specific antibodies from polyclonal antisera by adsorption on non-target antigens immobilized on nitrocellulose membranes. This "negative" purification method is simple and provides better immunoreagents than the blocking of nonspecific antibodies in solution or the enrichment of specific antibodies on nitrocellulose membranes. For routine applications, this method is quicker and cheaper than the purification protocols based on selective precipitations and affinity chromatography.     

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

Prevention of artifactual oxidation in determination of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction

A highly sensitive quantitative method based on LC-MS/MS was developed to directly measure 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) in crude DNA hydrolysates. With the use of isotopic internal standards and online solid-phase extraction (SPE), this method has overcome the artifactual response often observed during electrospray ionization by optimizing the washing conditions of online SPE to remove excess dG and allows 8-oxodG and dG to be accurately and simultaneously monitored by mass spectrometry. The detection limit of this method was estimated as 1.8 fmol for 8-oxodG. With this method, we further investigated the artifactual oxidation that occurred during concentration and purification of the DNA hydrolysates, commonly used before sample analysis. Our results demonstrated that drying under vacuum or purification with C18 cartridges led to a significant increase in the measured 8-oxodG by 6.8-30 8-oxodG/10(6) dG. The artifactual formation of 8-oxodG can be reduced only by adding desferrioxamine (DFO) and not 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). However, DFO still failed to offer complete protection against oxidation during DNA hydrolysate concentration and purification. Therefore, to effectively prevent the artifacts formed during workup, the simplest approach is to use a direct measurement method involving an online enrichment/purification technique as proposed in this study.     

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-μm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-μm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.     

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness¹. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)^2,3 and a C-terminal 10xHis tag⁴, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression⁵. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme⁶. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl₂ and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.     

A method for purifying the platelet membrane glycoprotein IIb-IIIa complex

A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) IIb and IIIa. This method produces an excellent yield and does not require the prior isolation of platelet membranes. Outdated platelets were washed and solubilized in Triton X-100. Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-Sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. Wheat germ agglutinin affinity chromatography was used to completely remove trace amounts of fibrinogen. The purified GP IIb and GP IIIa were analyzed by sucrose gradient sedimentation and found to consist of heterodimer complexes.     

Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.     

A simple and effective separation and purification procedure for DNA fragments using Dodecyltrimethylammonium bromide

In this work we describe a simple two step separation procedure for the separation and purification of short DNA fragments. The first step involves precipitating the DNA using the cationic surfactant dodecyltrimethylammonium bromide. Dodecyltrimethylammonium bromide, unlike cetyltrimethylammonium bromide will not precipitate DNA before complexation is complete thus providing a high purity DNA. The second step involves dissolution of the DNA-dodecyltrimethylammonium complex in 75% ethanol, followed by precipitation of the Sodium-DNA salt, by titrating in a salt solution. This method is particularly suited to purification of short fragments as it does not require high salt concentrations in the ethanol precipitation step, which can be damaging for short DNA. The ability of dodecyltrimethylammonium bromide to remove ethidium bromide from intercalation sites on the DNA is also discussed     

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6?×?His–TF–CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF–CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase–human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25?mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.     

Removal of the fermentation by-product succinyl L-tyrosine from the beta-lactamase inhibitor clavulanic acid using a molecularly imprinted polymer

Clavulanic acid is a beta-lactamase inhibitor used in therapeutic combinations with the penicillin-type antibiotics. During the fermentation leading to clavulanic acid, a succinyl L-tyrosine by-product is unavoidably formed. Occasionally, the amount of this by-product is found to be as high as 2% of the product even after standard purification operations. To further remove this impurity, we prepared a highly specific adsorbent for succinyl L-tyrosine with the molecular imprinting technique. This was performed by simultaneously using vinylbenzyl trimethylammonium chloride and methacrylic acid as the functional monomers. The imprinted polymer selectively bound succinyl L-tyrosine, and could be successfully used to remove this impurity at concentrations of less than 2% in the presence of clavulanic acid.     

中试规模纯化海洋芽孢杆菌源脂肽类化合物

本次研究旨在建立经济可行的海洋芽孢杆菌源脂肽类化合物的中试规模纯化工艺。对包括酸化沉淀、甲醇浸提、溶剂沉淀、盐析、萃取、硅胶柱层析和HZ806大孔树脂吸附工艺在内的可放大的成熟单元工艺进行反复试验,考察脂肽类化合物表面活性对单元工艺的影响。严格遵循以高收率为前提循序渐进逐步减少杂质的原则,组合上述单元工艺对目标产物进行提取和纯化,并最终获得高纯度脂肽样品。新工艺可从1 t海洋芽孢杆菌Bacillus marinus B-9987的发酵液中,以百克量级的规模制备87.51%–100%纯度的脂肽类化合物样品,收率81.73%。本研究首次实现了高纯度的海洋芽孢杆菌源脂肽类化合物的百克量级制备;允许发酵生产阶段使用天然培养基,缓解了脂肽中游发酵生产和下游大规模纯化之间的矛盾;且各单元工艺规避了脂肽类化合物水溶液的乳化起泡和不经济的大体积水溶液蒸发浓缩。新工艺实用可行,经济合理。     

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.     

Author index for volume 202

A procedure is described for a simple two-step purification of human liver propionyl-CoA carboxylase. The method is based on acid and carbon tetrachloride extraction to remove other biotin carboxylases followed by an 800-fold purification through biotin-pretreated, monomeric avidin-Sepharose 4B-CL with elution of active enzyme using a biotin gradient. The enzyme had a sedimentation coefficient of 17.4 S and polyacrylamide gel electrophoresis after reduction and alkylation revealed two nonidentical polypeptide chains of 75,000 and 60,000 M_r. The heavier chain was identified as the biotin-containing subunit by electrophoresis after avidin binding.     

层析法去除A群流脑多糖疫苗粗糖中杂蛋白

目的:建立用层析法去除脑脊髓膜炎球菌A群荚膜多糖样品中的杂蛋白的方法,以替代传统的苯酚抽提。方法:A群流脑多糖粗糖经过新型多模式离子交换填料Capto adhere,使用低盐上样高盐洗脱,洗脱的目的峰再经过Sephardex G-25凝胶过滤脱盐换成水,即为流脑多糖精糖原液。结果:采用上述方法制备的精制多糖样品中杂蛋白占固总的比例低于0.2%,符合WHO现行规程中的杂蛋白比例低于1%的要求,核酸、内毒素等生化指标均符合现行规程,整个层析过程的回收率大于65%。结论:层析法去除A群流脑多糖粗糖中的杂蛋白是有效的,比传统的苯酚抽提的方法更有利于环保,易于操作,易于放大。     

Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.