3- Instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.     

The effect of physiologically relevant additives on the rheological properties of concentrated Pluronic copolymer gels

The high concentration triblock copolymer poly(ethylene oxide)₉₉-poly(propylene oxide)₆₉-poly(ethylene oxide)₉₉ (Pluronic F127) aqueous solutions with the addition of different components commonly used in physiologically relevant applications were characterized by rheological measurements, differential scanning calorimetry (DSC) and small angle X-ray/neutron scattering. The sol-gel transition temperature, as well as the storage modulus of the F127 solution depend both on the concentration of polymer and of clay. Above the gel transition, the storage modulus of the solutions increased with clay concentration. Yield strain is independent of polymer and clay concentrations. Two different kinds of inorganic salts, sodium chloride (NaCl) and calcium chloride (CaCl₂) were added into the polymer and polymer-clay solutions. The sol-gel transition temperature decreased noticeably, but the storage modulus decreased only a small amount with increasing concentration of inorganic salts. Addition of salts to polymer-clay solutions resulted in precipitation of the clays which decreased the modulus. No effect on the mechanical properties was observed with the addition of common serum proteins. However, addition of 0.5-10% glucose decreased the transition temperature between 3° and 7°, without significantly affecting the modulus. The depression of the transition temperature by glucose was similar to that found with salts and indicated that the mechanism, namely competition for water, may be similar.     

用蛋白质工程技术提取和修饰南极假丝酵母脂肪酶B

从南极假丝酵母中提取的脂肪酶B,具有较高的对应异构体选择性,广泛应用在培养基中,作为不对称有机化学的生物催化剂。近年来,一系列组合蛋白质工程研究和扩展了南极假丝酵母脂肪酶的催化和物理特性。这些工程不但生产出了指定的催化剂,还有助于阐明酶的结构一作用关系,并且有助于说明这些酶的对应异构体选择性。进一步的研究将在工程与酶的关系方面展开讨论。     

Effect of alkali on the refunctionalization of soy protein by hydrothermal cooking

The effects of hydrothermal cooking (HTC) at alkaline conditions on refunctionalization of heat-denatured protein of extruded-expelled (EE) soy meals and on preparation of soy protein isolate (SPI) from EE soy meal were determined. Two HTC setups, flashing-out HTC (without holding period) and HTC with holding for 42 s at 154°C, were evaluated. Alkali (NaOH) addition dramatically enhanced the refunctionalization of EE meal having an initial protein dispersibility index of 35. The more alkali added, the more refunctionalization occurred. Extensive refunctionalization was achieved at 0.6 mmol alkali/g EE meal, and additional improvement was small with more alkali. For both HTC setups, the solids and protein yields of SPI from alkali-HTC-treated EE meals were significantly higher than those from HTC without alkali addition. The yield of protein as SPI increased from 40 to 82% after HTC treatment at 0.6 mmol alkali/g EE meal compared with no alkali addition. The emulsification capacities of SPI after alkali-HTC were similar to those from HTC without alkali. SPI from holding-tube HTC-treated EE meals had higher emulsification capacities than those prepared by flashing-out HTC.     

Exo-mechanism proximity-accelerated alkylations: investigations of linkers, electrophiles and surface mutations in engineered cyclophilin-cyclosporin systems

Investigations on the scope and utility of exo-mechanism proximity-accelerated reactions in engineered receptor-ligand systems are reported. We synthesized a series of electrophilic cyclosporin (CsA) derivatives by varying electrophiles and linker lengths, prepared a series of nucleophilic cysteine mutations on the surface of cyclophilin A (Cyp), and examined their reactivity and specificity in proximity-accelerated reactions. Acrylamide and epoxide electrophiles afforded useful reactivity and high specificity for alkylation of engineered receptors in Jurkat cell extracts. We found that remote cysteines (>17 A from the ligand) could be alkylated with useful rates under physiological conditions. The results from mutations of the receptor surface suggest that the dominant factors governing the rates of proximity-accelerated reactions are related to the local environment of the reactive group on the protein surface. This study defines several parameters affecting reactivity in exo-mechanism proximity-accelerated reactions and provides guidance for the design of experiments for biological investigations involving proximity-accelerated reactions.     

温敏性微凝胶对蛋白质和酶的吸附性能

用微乳液聚合方法以N-异丙基丙烯酰胺为单体合成了温敏性微凝胶聚N-异丙基丙烯酰胺(PNIPAM),研究了其对两种蛋白质和两种酶的吸附性能,测定了吸附等温线和温度对吸附量的影响。结果表明,微凝胶在低临界溶解温度(LCST)附近吸附蛋白质和酶的量有一突跃,例如在LCST前后,1 g纳米颗粒吸附的酪蛋白的质量分别为225 mg和415 mg;吸附的枯草杆菌蛋白酶的质量分别为12 000U/mg和27 500 U/mg。蛋白质和酶是通过物理吸附作用结合到PNIPAM微凝胶上,可以用调节温度的方法,来控制温敏微凝胶对蛋白质和酶的吸附与脱附。     

反胶团萃取蛋白质技术的新进展

反胶团液-液萃取技术具有条件温和、高选择性、易放大等优点,是一种应用前景极好的生化分离方法。本文介绍反胶团的性质、萃取原理,并着重分析了制约反胶团萃取技术实用化的各种因素及研究现状,评述其工业应用前景。     

利用耐酸霉菌噬酸柱霉从废纸酸水解产物中生产单细胞蛋白

在pH1的条件下,废纸通过噬酸柱霉生物转化成单细胞蛋白。废纸在4℃时用72%硫酸预处理,用水稀释至pH0.1时水解。为霉菌的生长产生足够的含糖基体。在水解产物中有97%的糖(葡萄糖、半乳糖、木糖、阿拉伯糖)转化为细胞物质。没有发现微生物污染。以糖的消耗为根据,在曝气培养条件下,噬酸柱霉的产率平均值和蛋白质平均含量实际上比经摇床培养的高。霉菌细胞的化学组成和必要的氨基酸和其他霉菌类似。     

A群链球菌四价重组蛋白的表达、纯化及其免疫原性

目的原核表达并纯化A群链球菌M1、3、6、18型四价重组蛋白,并检测其免疫原性。方法以克隆质粒pUC18-Strep4为模板,PCR法扩增四价重组蛋白基因,克隆至表达载体pQE30中,构建重组表达质粒,转化E.coilM15,筛选阳性克隆,IPTG诱导表达。采用Ni2+-NTA凝胶亲和层析纯化重组蛋白。以该蛋白免疫家兔,ELISA法检测血清抗体滴度;间接免疫荧光法(IFA)检测A群链球菌型特异性抗体;体外杀菌试验检测杀菌抗体活性。结果四价重组蛋白表达质粒pQE30-Strep4经双酶切和测序,证明构建正确。重组蛋白相对分子质量与预期相符,表达量约占菌体总蛋白的30%,为可溶性表达。纯化后蛋白纯度可达90%以上,并可诱导家兔产生高滴度的M1、3、6型特异性杀菌抗体。结论已成功表达了A群链球菌四价重组蛋白,纯化后的蛋白可诱导家兔产生针对A群链球菌M1、3、6三个血清型菌株的特异性杀菌抗体。     

人细小病毒B19的特性及其与血浆蛋白制品的相关性

人细小病毒B19(B19病毒)是一种直径约为20～25nm的无包膜单链线状DNA病毒,属于细小病毒家族。B19病毒对人类骨髓细胞,特别是红系祖细胞有高度趋向性,易感人群感染B19病毒可引起多种严重疾病,一般通过呼吸道和母婴垂直传播,但通过输注血液和血浆蛋白制品传播也非常普遍,已引起人们的广泛关注。本文对B19病毒的特性及其与血浆蛋白制品的相关性作一综述。