Expression and Purification of ZNF191（243-368） in Three Expression Systems

ZNF191 （243-368）, a new human zinc finger protein, probably relates to some hereditary diseases and cancers, To obtain adequate amount of ZNF191（243-368） for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase （GST）, and hexahistidine （6 × His） were used and compared. Among these systems, the expression level of ZNFI91（243-368） was increased in inclusion body system under a higher isopropylthio-β-D-galactoside （IPTG） concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.     

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of （ R, S ） -2-Octanol Acetate

To identify the desired hyperthermophilic variants within a mutant esterase library for the resolution of (R,S)-2-octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96.2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.     

Synthesis of Symmetric Diketones from Imidazolinium Salt and Bis-Grignard Reagents

In our earlier paper, we reported a convenient method for the synthesis of symmetricdiketones from benzimidazolium salts and his-Grignard reagents'. The yields of productobtained by the reported method are better then that of the former methods2' 3. Howeverthe method suffers from dramatic expensive material, such as o-phenyldiamine, whichmakes it not to be useful for large-scale preparation.In order to overcome this disadvantage and find a new method which can be utilizedin industry, we have e…     

Studies on the Interaction of Beryllon Ⅲ with Proteins by Voltammetric Technique and its Application

A new quantitative determination method of proteins using beryllon Ⅲ by voltammetric technique was developed in this paper. In pH 3.5 Britton-Robinson (B-R) buffer solution, beryllon Ⅲ can bind with human serum albumin (HSA) to form an electro-inactive supermolecular complex. Beryllon Ⅲ has a well-defined voltammetric reduction peak at -0.38 V (vs. SCE) and the addition of protein in this solution can cause the decrease of the reductive peak current. Based on the decrease of the reduction peak current, a new electrochemical method for the determination of HSA was established with linear range of 1.0-40.0 mg/L and the detection limit of 1.0 mg/L. This method is further applied to the determination of real sample of healthy human serum.     

Stability of proteins with multi-state unfolding behavior

A new model used to calculate the free energy change of protein unfolding is presented.In this model,proteins are considered to be composed of structural elements.The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method.If a protein consists of the same structural elements,its unfolding will displays a two-state process,and only the average structural element free energy change < △G 0 element(H2O)> can be measured.If protein consists of completely different structural elements,its unfolding will show a multi-state behavior.When a protein consists of n structural elements its unfolding will shows a(n+1)-state behavior.A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration,not to the start of each transition.The measured △G0 protein(H2 O) is the sum of the free energy change for each structural element.Using this new model,we can not only analyze the stability of various proteins with similar structure and similar molecular weight,which undergo multi-state unfolding processes,but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change < △G0 element(H2O)>.Although this method cannot completely provide the exact free energy of proteins,it is better than current methods.     

The preparation and fluorescence properties of europium nanoparticles

A new structured metallic nanomaterial of europium nanoparticle was prepared using tannic acid as the reductive agent,and nanoeuropium protein conjugates were synthesized by the method of lipoic acid modification on the surface of nanoparticle,which opens a new field of application of lanthanides in nanotechniques.Their properties were also characterized by UV-vis absorption spectroscopy,transmission electron microscopy (TEM),and fluorescence spectroscopy.The europium nanoparticle and its protein conjugates solution were stable and water-soluble.The fluorescence intensity of the composite europium nanoparticles was significantly increased in the presence of trace protein,and was linear proportional to the concentration of proteins under optimum conditions.According to this,a fluorimetric method for the determination of protein was developed in this paper.     

Synthesis of cis-(±)-2-Hydroxymethyl-5-(Cytosine-1'-yl)-1,3-Oxathiolane (BCH-189)

cis-BCH-189 are potent anti-HIV agents and have been synthesized from mannose1 galactose2 or glucose3 etc. Owing to so many synthetic steps, it is difficult and expensive to obtain a few grams. In this paper, we have designed new route and promoted the reaction conditions for the synthesis of this nucleoside cis- (+)- BCH-189 from cheap starting material via a four step route as shown in Scheme 1. By this method, a series of derivatives of title compound can be synthesized conviniently for…     

Catalytic enantioselective synthesis of β-amino alcohols by nitrene insertion

Chiral β-amino alcohols are important building blocks for the synthesis of drugs, natural products, chiral auxiliaries, chiral ligands and chiral organocatalysts. The catalytic asymmetric β-amination of alcohols offers a direct strategy to access this class of molecules. Herein, we report a general intramolecular C(sp³)–H nitrene insertion method for the synthesis of chiral oxazolidin-2-ones as precursors of chiral β-amino alcohols. Specifically, the ring-closing C(sp³)–H amination of N-benzoyloxycarbamates with 2 mol% of a chiral ruthenium catalyst provides cyclic carbamates in up to 99% yield and with up to 99% ee.The method is applicable to benzylic, allylic, and propargylic C–H bonds and can even be applied to completely non-activated C(sp³)–H bonds, although with somewhat reduced yields and stereoselectivities. The obtained cyclic carbamates can subsequently be hydrolyzed to obtain chiral β-amino alcohols. The method is very practical as the catalyst can be easily synthesized on a gram scale and can be recycled after the reaction for further use. The synthetic value of the new method is demonstrated with the asymmetric synthesis of a chiral oxazolidin-2-one as intermediate for the synthesis of the natural product aurantioclavine and chiral β-amino alcohols that are intermediates for the synthesis of chiral amino acids, indane-derived chiral Box-ligands, and the natural products dihydrohamacanthin A and dragmacidin A.     

Interaction of zinc finger ZNF191(243—368) and its I323 W and R327W mutants with oligonucleotides by florescence spectrum

Two mutants of the zinc finger protein, ZNF191 (243–368) I323W and R327W, are successfully obtained by site-directed mutagenesis. The fluorescence spectrum is used to study the interaction between these two mutants and the oligonucleotides. The influence of the mutation on the interaction has been studied using ethidium bromide (EB) as the fluorescence probe. The binding constants of the I323W-DNA and R327W-DNA have been calculated and the possible binding models have been discussed.     

Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil

Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(UV) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as Cu(2+), Ni(2+), or Zn(2+). The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third alpha-helices of IZ-HH. Escherichia coli cells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the Cu(2+) and Zn(2+) ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the (191)cpGFP(190) domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.     

Liquid-liquid coexistence surface for lysozyme: role of salt type and salt concentration

The liquid-liquid phase separation curves for lysozyme in a salt solution are known to depend on salt type and salt concentration. For the case of monovalent cations, the cloud point temperature typically increases with increasing salt concentration, for fixed lysozyme concentration. For the case of divalent cations, however, a maximum in the cloud point temperature is observed that has been interpreted as being due to ion binding to the protein surface and subsequent water structuring. In this paper, we use a simple square well model due to Grigsby et al. (Biophys. Chem. 2001, 91, 231-243), whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces are shown as a function of temperature, salt concentration, and protein concentration for two typical salts, NaCl and MgCl2. These surfaces are calculated using the results of a single standard Monte Carlo simulation and a simple scaling argument and are in reasonably good agreement with known experimental results.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.