Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by β-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).     

Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers

Lycopene has a large number of geometric isomers caused by E/Z isomerization at arbitrary sites within the 11 conjugated double bonds, offering varying characteristics related to features such as antioxidant capacity and bioavailability. However, the geometric structures of only a few lycopene Z-isomers have been thoroughly identified from natural sources. In this study, seven multi-Z-isomers of lycopene, (9Z,13′Z)-, (5Z,13Z,9′Z)-, (9Z,9′Z)-, (5Z,13′Z)-, (5Z,9′Z)-, (5Z,9Z,5′Z)-, and (5Z,9Z)-lycopene, were obtained from tomato samples by thermal isomerization, and then isolated by elaborate chromatography, and fully assigned using proton nuclear magnetic resonance. Moreover, the theoretically preferred pathway from (all-E)-lycopene to di-Z-isomers was examined with a computational approach using a Gaussian program. Fine-tuning of the HPLC separation conditions led to the discovery of novel multi-Z-isomers, and whose formation was supported by advanced theoretical calculations.     

小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

在前期研究中分离到噬碳酸盐小球藻（Chlorella sp.X1）的亲环蛋白基因CsCyp1A（登录号：KY207381）,编码CsCyp1A蛋白是否具有肽酰脯氨酰顺反异构酶（PPIase）活性功能是进一步研究它参与小球藻耐盐生物学过程的基础。通过His标签的pQE-30-CsCyp1A原核蛋白表达载体,经IPTG诱导它的E.coli M15菌株表达融合蛋白,Nitra-柱纯化后获得纯化的30 kDa左右的His-CsCyp1A融合蛋白质,Western杂交检测到HisCsCyp1A蛋白的杂交信号。酶活性分析显示,HisCsCyp1A融合蛋白中生色基团的产生速度明显快于对照,表明CsCyp1A蛋白可以催化底物N-succinyl-Xaa-Pro-Phe-p-nitroanilide中Xaa-Pro肽键的顺反折叠,说明纯化的CsCyp1A蛋白具有PPIase活性。典型的亲环蛋白家族成员具有肽酰脯氨酰顺反异构酶（PPIase）活性,参与折叠和转运、信号转导、免疫调节、细胞凋亡等生物学过程。真空渗入法侵染转化的拟南芥在35S启动子驱动下超表达CsCyp1A基因,耐盐性研究表明它提高了过表达株系对NaCl胁迫的耐性,揭示小球藻CsCyp1A基因的抗盐性功能,它将成为抗逆育种的基因资源。本研究也为探索小球藻亲环蛋白A的抗盐碱胁迫生物学作用和分子育种奠定了基础。     

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.     

Green‐lighting green fluorescent protein: Faster and more efficient folding by eliminating a cis–trans peptide isomerization event

Wild‐type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis–trans peptide bond isomerization. The only conserved cis‐peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X‐P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans‐peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.     

Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501

A gene (thaI) corresponding to l-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the l-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60°C at pH 8 for 30 min of reaction. Zn²⁺ and Ni²⁺ inactivated the catalytic activity of ThaI, 5 mM Mn²⁺ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from d-galactose to d-tagatose was obtained by recombinant ThaI at 60°C over 3 d.     

塔格糖-4-异构酶的新酶挖掘及酶学性质研究

d-塔格糖是一种己酮糖,在自然界中天然存在但含量极少,具有降血糖、抗龋齿、改善肠道菌群、促进血液循环和抗衰等作用,可广泛应用于食品、医药和化妆品行业。目前酶催化半乳糖生成塔格糖仅需一步反应,简单易行,但半乳糖较高的成本限制了这一方法的工业应用。果糖是塔格糖的同分异构体,价格低廉,来源稳定,可通过4位差向异构化一步获得塔格糖,是产塔格糖的理想底物。【目的】因此挖掘新的塔格糖-4-差向异构酶对塔格糖的工业生产具有重要意义。【方法】本文通过基因挖掘的手段发现了热袍菌(Thermotogae bacterium)来源的未知功能蛋白(命名为HCZ0)具有塔格糖-4-差向异构酶活性,进而完成了HCZ0的异源表达、纯化及酶学性质表征。【结果】确定HCZ0表观分子量约为57.9 kDa,比活力为0.9 U/mg,最适反应温度和pH值分别为70 ℃和9.0,最适金属离子为Ni²⁺,最适金属离子浓度为2 mmol/L,60、70和80 ℃半衰期分别为180、67和9 h。最适条件下,HCZ0催化200 g/L果糖在2 h内可产生28 g/L塔格糖。【结论】本研究中,HCZ0是碱性高温酶,且具有较好的热稳定性,这些特征在以后的理论研究及工业生产中具有一定的科学价值。     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.