Formation of giant protein vesicles by a lipid cosolvent method

This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent-driven fusion of large vesicles (0.1-0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.     

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual‐Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10^?8 cm² s^?1 for the photoreceptor and with D=4.1×10^?8 cm² s^?1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.     

Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells

Hemoproteins on their metal: We report a novel strategy for the reconstitution of hemoproteins with non-natural metal complexes; simple addition of manganese and ruthenium porphyrin to E. coli cells immediately prior to homogenization yields the reconstituted proteins. We believe that this simple approach could become a standard reconstitution method for hemoproteins.     

课程重组与模块教学模式的探讨

从职业教育的培养方向、模式要求等方面，阐述了课程重组，即课程整合与模块化教学在高等职业教育中的地位、作用。对机电技术应用专业的课程整合、模块化教学模式从整合的依据、整合的要求、模块的划分、专业能力要求的设定、师资、设备配备、教材的编写等方面进行了探讨。     

重组蛹虫草超氧化物岐化酶脱辅基蛋白的表达、纯化及其活性重建

目的表达并纯化重组蛹虫草超氧化物歧化酶脱辅基蛋白,并考查各种金属离子对其活性重建的作用。方法用不含Cu2+和Zn2+的培养基培养重组菌株,并表达重组蛋白,采用DEAE-FF和CM-52进行纯化;在脱辅基蛋白溶液中加入Cu2+、Mn2+、Mg2+、Ni2+或Ag+,测定SOD酶活力的变化。结果蛹虫草脱辅基Cu,Zn-SOD在不含Cu2+和Zn2+的培养基中得到表达,纯化后的样品经SDS-PAGE分析,在相对分子质量17000处出现单一条带,经活性电泳分析无酶活力;脱辅基蛋白活性重建的最适宜Cu2+浓度为0.005mmol/L,但其紫外吸收图谱与天然SOD不同;0.1mmol/L的Mn2+、Mg2+、Ni2+、Ag+重建的酶活力远低于Cu2+重建的cm-SOD的酶活力。结论已成功表达并纯化了蛹虫草脱辅基Cu,Zn-SOD,各种金属离子只能有限地重建脱辅基Cu,Zn-SOD的活性。     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Formation kinetics and stability of surfactant vesicles

A three-component phase diagram of a system containing water, dimethyl isosorbide and Laureth 4 (Brij^® 30), a commercial surfactant, was determined, and the kinetics of vesicle formation from dilution with water of the hydrotrope solution was studied using a stop-flow process in conjunction with light-scattering determinations. The phase diagram consisted of a large microemulsion phase and a lamellar liquid crystalline region. Results from the stop-flow/light-scattering determinations were tentatively interpreted using the Aniansson-Kahlweit-Zana theory of micellar relaxation for a system close to equilibrium. The interpretation indicated the vesicles to be formed by monomolecular buildup for surfactant concentrations less than 5%, while for vesicles formed at greater concentrations an agglomeration of vesicle fractions appeared more reasonable.     

Interaction of vesicles: adhesion, fusion and multicompartment systems formation

In this review, interactions of selected vesicles, induced either by molecular recognition or by electrostatic interactions, for the simulation of cell-cell and cell-drug interaction processes are discussed. In order of increasing complexity, examples of vesicles adhesion are presented at first, followed by recognition experiments in which fusion takes place. This differentiation in behavior was primarily attributed to the structural features of interacting vesicular pairs primarily affected by concentration and lateral phase separation of the anchored recognizable groups. In certain cases, fusion is accompanied by multicompartmentalization of the obtained aggregates. In connection with the formation of these multicompartment systems, it was proposed that an analogous mechanism could be operating in the evolution of eukaryotes during the symbiosis of prokaryotes.     

Evaluation of in vitro stability of small unilamellar vesicles coated with collagen and chitosan

To improve the in vitro stability of small unilamellar vesicles (SUV), the permeability of SUV, made from soyabean phosphatidylcholine (PC) and coated with collagen and chitosan, was studied using 5(6)-carboxyfluorescein (5(6)-CF) as a fluorescence probe. The results showed that the coating with a collagen/PC weight ratio of 2, or a chitosan/PC weight ratio of 8, significantly decreased the permeability of liposomal membranes. In addition, the fluorescence polarization method was used to study the influence of a coating with collagen and chitosan at the above ratios on the fluidity of liposomal membranes, employing an intramolecular charge transfer compound, a 3-methoxy-4′-N ,N-dimethylaminoflavone derivative (DMMF), as a fluorescence probe. The fluidity of a liposomal membrane coated with either of the two macromolecules showed no obvious changes, indicating that SUV coated with collagen and chitosan at appropriate weight ratios, can significantly improve the in vitro stability of liposomal membranes without disturbing their fluidity. © 1999 Society of Chemical Industry     

Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

From its start as a small‐scale in vitro system to study fundamental translation processes, cell‐free protein synthesis quickly rose to become a potent platform for the high‐yield production of proteins. In contrast to classical in vivo protein expression, cell‐free systems do not need time‐consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high‐throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell‐free systems are of enormous interest. The modification of the genetic code to incorporate non‐canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell‐free projects. Many sophisticated cell‐free systems for manifold applications have been established. This review describes the recent advances in cell‐free protein synthesis and details the expanding applications in this field.     

Structure and crystal growth approach of poly(alkylacrylate) myelinic vesicles by electron microscopy

Alkylacrylate copolymers

form a myelinic phase showing vesicles $\text{～1000}\text{A}\text{?}$ in diameter and a minimum thickness of 30 Å. Electron microscope and diffraction patterns show that the membrane is a monolayer of the hexagonal stacking of the C₂₂ alkyl chains. A structural model is proposed showing how the alkylchains are connected by the principal polymer chain. The growth mechanism of such vesicles is discussed.     

Recovery and utilisation of by-product proteins of the meat industry

The methods available for the recovery and utilisation of proteins from blood and offals are reviewed: special reference is made to the use of controlled enzymic hydrolysis. The texturisation of these proteins into meat-like analogues, by fibre spinning or thermoplastic extrusion, is discussed.     

Experimental Aspects of Colloidal Interactions in Mixed Systems of Liposome and Inorganic Nanoparticle and Their Applications

In the past few years, growing attention has been devoted to the study of the interactions taking place in mixed systems of phospholipid membranes (for instance in the form of vesicles) and hard nanoparticles (NPs). In this context liposomes (vesicles) may serve as versatile carriers or as a model system for biological membranes. Research on these systems has led to the observation of novel hybrid structures whose morphology strongly depends on the charge, composition and size of the interacting colloidal species as well as on the nature (pH, ionic strength) of their dispersing medium. A central role is played by the phase behaviour of phospholipid bilayers which have a tremendous influence on the liposome properties. Another central aspect is the incorporation of nanoparticles into vesicles, which is intimately linked to the conditions required for transporting a nanoparticle through a membrane. Herein, we review recent progress made on the investigations of the interactions in liposome/nanoparticle systems focusing on the particularly interesting structures that are formed in these hybrid systems as well as their potential applications.