Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

人睫状神经营养因子的原核表达,纯化及其生物效应

人睫状神经营养因子（ｈＣＮＴＦ）克隆入ｐＢＶ２２０中，在ＤＨ５α菌株中表达，重组蛋白以包含体的形式存在，表达量为菌体总蛋白的５０％左右。经比较发现用２ｍｏｌ／Ｌ脲洗涤包含体可溶解大量可溶性细菌蛋白，且包含体损失较小。在高浓度变性剂条件下进行ｓｅｐｈａｒｃｙｌＳ－２００凝胶过滤，解决了纯化中ｈＣＮＴＦ易聚合的问题，在低浓度变性剂条件下进行ＤＥＡＥ离子交换，有利于蛋白活性的保持。经两步纯化后得到均一性ｈＣＮＴＦ，纯度达９５％以上。在自然状态下使ｈＣＮＴＦ复性。纯化复性后的ｈＣＮＴＦ对无血清培养的鸡胚背根节神经元和脊髓腹角运动神经元有明显的维持存活和促进生长发育的生物效应。     

Similarities and differences in the properties of multiple isoforms of maize endosperm casein kinase I (CK-I)

Two novel type I casein kinases named CK-1B and CK-1C have been purified from maize endosperm (three weeks after anthesis) by a six step procedure involving ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-75, Heparin-sepharose, and ATP-agarose chromatography. The catalytic subunits of both enzymes were identified as a 35-37 kDa polypeptide doublet by in situ phosphorylation after SDS/PAGE in active casein gel. Both enzymes required 5-10 mmol · L⁻¹ Mg²⁺ for maximal activity, could utilize only ATP as phosphate donor, were insensitive to heparin, were not autophosphorylated, had a pH optimum at pH 7 to 8.5, and exclusively phosphorylated acidic proteins (casein, phosvitin). Regarding the enzyme differences, their properties were as follows: a) CK-1B could bind on ATP-agarose affinity column, while CK-1C could not; b) the activity of CK-1C was strongly stimulated at low concentrations (1 mmol/L) of spermine, while that of CK-1B was inhibited; c) CK-1B and CK-1C Km values for ATP were 11 μmol · L⁻¹ and 26 μmol · L⁻¹, respectively; d) Mg²⁺ could substituted by Mn²⁺ in the CK-1B catalytic activity (by about 80 percnt;); e) CK-1B phosphorylated serine, while CK-1C both serine and threonine on casein. The combination of these results with those from Babatsikos and Yupsanis (2000) brings the number of investigated maize endosperm CK-I isoforms to three (CK-1B, CK-1C, and CK-1E). This is the first biochemical approach demonstrating that multiple isoforms of CK-I casein kinases are present in the same plant tissue.     

Purification of three γ-chains with different molecular weights from normal human plasma fibrinogen

Three forms of the normal human plasma fibrinogen γ-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique γ-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Aα, Bβ and smallest γ-chain (γ₅₀) eluted at progressively higher ionic strengths, but the elution positions of Aα, Bβ and γ₅₀ chains were identifcal for fibrinogen from each of the three different chromatographic fractions. The unique γ chain of fibrinogen in the second chromatographic peak (γ₅₅) eluted at an ionic strength higher than that of the γ₅₀ chain, while the largest γ-chain (γ_57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the γ-chains unique to them, suggesting that the γ-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three γ-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD_260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.     

Salt-induced phase separation can effectively remove the acetonitrile from the protein sample after the preparative RP-HPLC

Acetonitrile (ACN)–water system is one of the most commonly used mobile phases in practical reverse-phase high-performance liquid chromatography (RP-HPLC). However, a higher concentration of ACN (normally greater than 60% (v/v)) is required to elute the target protein from the RP-HPLC column in which, further steps to remove the ACN from the protein samples are demanded. It has been demonstrated that the phase separation occurring under the sub-zero temperature could easily remove the majority of ACN from the effluent of RP-HPLC. Recently, we found that the comparable phase separation could be achieved by adding a small amount of proper salts, such as K₂HPO₄ and KH₂PO₄, and the phase separation could take place effectively at 4 °C where the protein-purification processes were usually carried out. In addition, the pH value of the solution could be maintained properly by using potassium phosphate buffer (pH 7.0). With an optimized condition for this salt-induced phase separation, we demonstrated that greater than 60% of ACN could be easily removed; on the other hand, more than 90% of water-soluble protein could be successfully recovered within five hours.     

Lipase of Pseudomonas cepacia for biotechnological purposes: purification,crystallization and characterization

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4°C in the eluent, containing 58–69% 2-propanol. The yield of the lipase was 87–100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-Page and capillary zone electrophoresis to be ≥ 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect of substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.