Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCI) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disul-fide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with re-versed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

Characters of the Plateau of Methanol Increment in Frontal Analysis in Reversed Phase Liquid Chromatography

With insulin methanol-water,and the ion-pairing agent,hydrochloric acid and trifluroacetic acid(TEA),the character of the first plateau(FP)on the elution curve of frontal analysis in reversed phase liquid chromatography(RPLC)was investigated by on-line UV-spectrometry and identified with nuclear magnetic resonance(NMR) spectrometry and mass spectrometry.The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis(FA).When the insulin concentration was limited to a certain range,the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten,The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components.The displaced methanol was quantitatively determined by NMR and on-line UV spectrometries.TFA with high UV absorbance can not be used as an ionpairing agent for the investigation of the FP in RPLC,but if can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC.A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC.From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC,while solute can penetrate into it in FA of RPLC.The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.     

STUDY ON AB-CROSSLINKED POLYMERS Ⅰ. THE SYNTHESIS AND MECHANICAL PROPERTIES OF AB-CROSSLINKED POLYMERS BASED. ON PU/PS

The AB-crosslinked polymers (i.e. ABCP) with polystyrene as chain A and vinyl group blocked prepolymers of polyurethanes (PU) as chain B were synthesized and studied. The results of dynamic mechanical spectrometry (DMS) show that the compatibility between the components A and B can be improved greatly through chemical crosslinking during the formation of ABCPs. This effect is especially pronounced when short chain prepolymers is chosen as one of the components. It is apparent that the degree of crosslinking between the two components plays a major role in determining their compatibility. Copolymerizafion of styrene with maleic anhydride in chain A can improve the compatibility and broaden the damping temperature range. Mechanical properties of the sythesized ABCPs were also studied.     

Effects of Various Dopants on Solitons in Polyacetylene

The effects of various dopants on solitons in polyacetylene were studied by using CNDO/2 level semiempirical quantum chemical method. The width of solitons is reduced when dopant is present, and the charge density wave(CDW) is further gathered on the carbon atom in soliton center. The effects of p-type of dopants are greater than those of n-type of ones. The charge transfer in doped polyacetylene can be achieved by the propagation of CDW along the chain. The conductivity of doped polyacetylene is proportional to the quantity of charge transfer between dopant and polyacetylene chain.     

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.     

The Important Roles of Water in Protein Folding: an Approach by Single Molecule Force Spectroscopy

The single-chain elasticity of a completely unfolded protein ((I27)8,modules of human cardiac titin) is studied in different liquid environments by the atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS).The experimental results show that there is a clear deviation between the force curves obtained in the aqueous and nonaqueous environments.Such a deviation can be attributed to the additional energy consumed by the rearrangement of the bound water molecules around the chain of the completely unfolded (I27)8 chain upon stretching in aqueous solution,which is very similar to the partial dehydration process from a denatured/unfolded to a native/folded protein.Through the analysis of the free energy changes involved in protein folding,we conclude that it is due to the weak disturbance of water molecules and the special backbone structures of proteins that the self-assembly of proteins can be achieved in physiological conditions.We speculate that water is likely to be an important criterion for the selection of self-assembling macromolecules in the prebiotic chemical evolution.     

Simultaneous Separation and Determination of Four Bioactive Constituents in Traditional Chinese Medicinal Tablet Xinkeshu by HPLC-DAD

Introduction In recent years there has been a renaissance of inter-est in use of traditional Chinese medicinal preparation (TCMP) because of the realization that traditional Chi-nese medicine can act in a synergistic manner in the human body, and can provide unique therapeutic prop-erties with minimal or no undesirable side-effects.1 The modernization research on TCMP is in process in China, and the first step is to establish simple and reliable ana-lytical technologies and methodologies f…     

REFINED CRYSTAL STRUCTURE OF INSULIN AT 1.8A RESOLUTION——HYDROGEN BONDS AND BOUND WATER

The hydrogen bonds in insulin fall into three cases: the helical hydrogen bonds in α- or 3_(10)helices, the non-helical one formed by polar groups of insulin itself, and the hydrogen bondsformed between insulin and water. By using the information obtained, the results of a seriesof biochemical investigations on insulin analogs related to B-chain C-terminal peptide can beinterpreted and it can also be inferred that the complex behaviours of the aggregation ofinsulin may play a protective role for the unique conformation of the molecule. Water structure also appears in the refined model. About one third of the water in anasymmetric unit is hydrogen-bonded to insulin molecules or each other, which are referred toas bound water. The polar and charged groups of insulin all show the tendencies to bind towater molecules as many as possible, which is a significant factor for the stabilization of theunique conformation of the molecule. The binding way of water molecules to insulin mole-cules is also analysed.     

Polarographic immunoassay coupled with catalysis of non-radioactive multiple iodine label

A now polarographic immunoassay was developed In this assay,human serum albumin (HSA) as the model antigen was covalently labeled with organic compound erythrosin B(EB) containing four non-radioactive iodides through Ⅰ step chemical reaction The labeling procedure is simple and the conditions needed are moderate.The molar labeling ratio of KB HSA was 12 Ⅰ The content of iodine in the conjugate obtained by the proposed procedure is ninth higher than that by the other existing methods.A heterogeneous competitive immunoassay was established by compling the catalysis of the conjugate to substrate As(Ⅲ)-Ce(Ⅳ) reaction with the linear-sweep polarographic detec-tion of As(Ⅲ) amount HSA can be determined in the HSA concentration range from 1 to 200μg/mL,with the de-tection hum of 0 66μg/ml.     

Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin's aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.     

用于蛋白同时复性及纯化的制备型装置中流动相用量的优化研究

 在生物大分子的高效液相色谱分离中 ,由计量置换保留模型可得出生物大分子在色谱柱上的保留行为取决于流动相中置换剂的浓度的结论。据此提出了用于蛋白同时复性及纯化的制备型装置 (USRPP)中最小流动相用量的估算公式 ,并进一步得出在保持最小流动相用量不变的条件下 ,改变流动相流速和线性梯度时间几乎不影响制备型USRPP分离蛋白的分离度和复性效率的结论。该结论与实验结果一致。     

用于变性蛋白质复性及同时纯化的简易型色谱柱的研制及其应用

研制出一种简易型色谱柱并通过装填大颗粒的疏水色谱填料对色谱柱的性能进行了考察,该柱的外形和操作如同传统的液相色谱柱,但它却在生物大分子分离方面有着和高效液相色谱相似的分辨率。另外,只要给该柱装填合适的固定相,比如疏水相互作用色谱固定相,便可用于蛋白质的复性及同时纯化。实验考察了该色谱柱的结构、操作和性能,包括柱压、柱寿命及对蛋白质的分辨率等。以溶菌酶为模型蛋白质,实验测得其在初始浓度为50.0 g/L时的质量回收率和活性回收率分别为(96.6±1.3)%和(101.1±6.0)%。这种简易型色谱柱价格低廉     

疏水作用色谱法同时纯化及复性基因重组人干扰素-α

 使用高效疏水作用色谱直接从大肠杆菌表达的基因重组人干扰素 α(rhIFN α)包涵体的裂解液中纯化了rhIFN α ,并在纯化的同时获得了高的复性效率 ,使复性和纯化一步完成 ,大大地简化了操作步骤。用凝胶排阻色谱对该法纯化的rhIFN α进行了纯度测定 ,纯度达到 95 %以上。该法的活性回收率分别比稀释法和透析法高 1 0倍和 1 6倍。     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to a-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

Theinvestigationofproteinrenaturation,orrefoldinghasbecomeaveryhotpointinbothliquidchromatography(LC)andlifesciencefields.Besidesusualdilutionanddialysismethods,manynewmethodsconcernproteinrenaturationhavebeenpresented,suchasreversemicelles1,chaperones2,polyethyleneglycol(PEG)3,surfactant4andantibodies5etc.Oneoftheauthorssuggestedhighperformancehydrophobicinter-actionchromatography(HPHIC)tobeanewtoolforproteinrenaturation6.Areviewonproteinrenaturationwithliquidchromatography(LC)wasrecentl…     

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to α-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

The contributions of tetrahydrofurfuryl alcohol(THFA) and polyethylene glyco(PEG) to the renature efficiency of α-chymotrypsin were investigated and compared with each other,The maximum increments of bioactivity recovery of α-Chy were found to be 25.1% for THFA,10.4% for PEG,respectively.The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of α-Chy in high performance hydrophobic interaction chromatography(HPHIC) than that containing PEG,when the concentration of THFA was 3.2%,the bioactivity recovery of α-Chy is the highest.     

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。     

Solubilization and Refolding with Simultaneous Purification of Recombinant Human Stem Cell Factor

Recombinant human stem cell factor (rhSCF) was solubilized and renatured from inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the solubilization of rhSCF inclusion bodies was investigated; the results indicate that the solubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution employed, and low concentration of urea can drastically improve the solubilization of rhSCF when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with simultaneous purification by ion exchange chromatography (IEC), with a specific activity of 7.8 × 10⁵ IU·mg⁻¹, a purity of 96.3%, and a mass recovery of 43.0%. The presented experimental results show that rhSCF solubilized by high pH solution containing low concentration of urea is easier to be renatured than that solubilized by high concentration of urea, and the IEC refolding method was more efficient than dilution refolding and dialysis refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.     

溶液中变性剂浓度对蛋白溶菌酶复性的影响

Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n（m1 -m2） and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography.     

源于大肠杆菌蛋白的表达、液相色谱复性与纯化新进展

对近两年来源于大肠杆菌（Escherichia coli,E．coli）的蛋白表达和用蛋白折叠液相色谱（protein folding liquid chromatography,PFLC）法对所形成的包涵体目标蛋白的复性并同时纯化的新近发展做了简要的介绍和评述．PFLC法用于包涵体蛋白分离、纯化很广,其特点是除了在色谱柱上将目标蛋白与其他组分分开,还同时要在色谱柱上进行包涵体蛋白折叠．可以说,现代生物技术中所用的大多数有价值蛋白产品的制备仍然有赖于不同机理的液相色谱（Lc）法．而用PFLC法对源于E．coli的蛋白的制备方法更具可塑性和容易达到规模化,其生成本可以成倍地降低．该文主要内容包括了E．coli蛋白的表达及样品前处理、PFLC的实用范围、PFLC的优化、PFLC中的新技术、新设备和新方法、PFLC的分子学机理、应用事例及对未来的展望．