Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation).     

一种基于形成杂多核络合物的荧光增敏效应的研究 Ⅲ.锰(Ⅱ)、钴(Ⅱ)的混合荧光增敏及其连续测定

本文研究了Mn~(2+)和Co~(2+)对7-(8-羟基-3,6-二磺基萘偶氮)-8-羟基喹啉-5-磺酸-硼砂反应体系的混合荧光增敏作用。实验条件下,荧光增敏强度满足线性加和关系的范围是:Mn~(2+)浓度0～2.9×10~(-7)mol/L;Co~(2+)浓度0～8.8×10~(-7)mol/L;总浓度不超过1.0×10~(-6)mol/L。检出限量为Mn~(2+)4.5×10~(-9)mol/L和Co~(2+)1.4×10~(-8)mol/L。实现了Mn~(2+)和Co~(2+)的连续测定。     

Signaling molecularly imprinted polymers: molecular recognition-based sensing materials

Molecular imprinting is a template polymerization technique that can easily provide synthetic polymers capable of molecular recognition for given target molecules. In addition to their highly specific recognition ability, we are attempting to introduce signaling functions to molecularly imprinted polymers, enabling them to respond according to specific binding events. Some of our work regarding such signaling molecularly imprinted polymers is presented here, including molecularly imprinted polymers that induce spectral shifts of target compounds because of binding. Such compounds include hydrogen-bonding-based fluorescent imprinted polymers and metalloporphyrin-based signaling molecularly imprinted polymers.     

Purification of recombinant green fluorescent protein by three-phase partitioning

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.     

Quantitative study on La~(3+) influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+uptake is obviously stimulated by pre-treating the cells with ouabain and by removing extracellular Na+, and intracellular La3+concentration ([La3+]i) is directly proportional to its extracellular concentration ([La3+]o). But when [La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum [La3+], is 1.5×10-12 mol · L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 μmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ion     

微波溶样肉桂基荧光酮和表面活性剂连续荧光测定葡萄酒中铁和钴

观察到肉桂基荧光酮(略作CF)在pH≈8与Fc(Ⅲ)配位后荧光增强,CPB的加入使荧光更强,络合物与试剂荧光强度之比F_(FeR)/F_R随0～0.01μg/ml Fe(Ⅲ)线性变化,λ_(ex)=410nm,λ_(ex)=480nm;而CF在pH≈10与Co(Ⅲ)配位后荧光却减弱,乳化剂OP存在时使试剂荧光增强,若以荧光熄灭Co(Ⅲ)灵敏度便提高,试剂与络合物荧光强度之比F_R/F_(CoR)对0～0.016 μg/ml Co(Ⅲ)呈直线关系,λ_(ex)=300nm,L_(em)=352nm。控制化学条件和激发、发射波长,可不经分离测定葡萄酒(微波溶样)中铁和钴。     

A High Sensitivity Fluorescent Chemo-sensory System Based on β-Cyclodextrin Dimer Modified with Dansyl Moieties

-Cyclodextrin dimer linked with ethylenediamine at the upper rim of the cyclodextrin has been synthesized and then modified with two dansyl moieties inthe presence of N,N'-dicyclohexylcarbodiimide. The sensing ability and bindingproperty of the title compound were investigated for steroids and terpenoids. Thefluorescence intensity of this dimer was decreased when a host–guest complex was formed. The value I/I⁰, where I⁰ and I are fluorescence intensitiesin the absence and presence of a guest and I is I⁰- I, was used as a parameter of sensitivity. This host exhibited a much higher sensitivity and selective molecular recognition ability for bile acids such as ursodeoxycholic acid andchenodeoxycholic acid and terpenoids such as (-)-borneol than the dansyl-modifiedcyclodextrins reported previously including -cyclodextrin dimer. The behaviors of the appended moieties of the host during the formation of host–guest complexes were studied using induced circular dichroism (ICD) and fluorescence spectra. The ICD intensityof this dimer was decreased on accommodation of a guest and this spectral pattern of the title dimer was opposite to that of bis dansyl-modified -cyclodextrin monomer. Theguest-induced variations in the fluorescence and ICD intensities suggest that this dimer formed a 1 : 1 host–guest complex and the appended moieties act as a hydrophobic cap.     

Advances of Chromatography in Analysis of Complex Samples

The separation and identification of complex samples is an urgent task that analytical chemistry has to face. Chromatography, as an important analysis technique, has been widely applied to many fields, such as life science, environment, medicine, food and petrochemical engineering.Recently, with the progress of science and tech-     

Determining PPARγ-ligand binding affinity using fluorescent assay with cis-parinaric acid as a probe

Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (Kd and Ki) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, Kd value of a probe can be obtained by binding assay, and Ki value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R]t), ligand([L]t) and probe([P]t) are required for the data fitting. In this work, Ki values of some typical ligands of PPARγ were successfully determined by use of our equations, among which the Ki value of PPARγ-LY171883 was reported for the first time.     

新型酰腙类荧光探针的合成及其对Al(Ⅲ)的传感性质

合成了两个酰腙类荧光探针1和2,在DMSO-H_2O(7∶3,体积比)体系中,两者分别在478 nm和460 nm处对Al~(3+)具有较好的荧光选择识别作用。Job's法、核磁滴定、质谱分析表明,探针1和2与Al~(3+)的配位比均为1∶2,且对Al~(3+)的检出限分别为9.58×10~(-8) mol/L和6.52×10~(-8) mol/L。同时提出了探针1和2对Al~(3+)的荧光传感机理。实际应用研究表明,探针1和2可用于河水和自来水中一定浓度范围内Al~(3+)的检测。