凝乳酶原Cys45-Cys50二硫键功能的研究

凝乳酶原突变体Cys45Asp/Cys50Ser包含体溶解所需的温度、时间、pH条件均与野生型一样,而且其氧化再折叠行为与野生型类似,在同样的复性条件下最终都能获得正确折叠可活化的分子.这与缺失Cys250-Cys283二硫键的突变体大不相同,说明Cys45-Cys50二硫键对凝乳酶原正确折叠的贡献小于Cys250-Cys283二硫键,但这对二硫键的缺失使假凝乳酶的热稳定性显著下降,说明此二硫键在稳定酶的空间构象中起重要作用、另外,突变体Cys45Asp/Cys50Ser假凝乳酶的水解蛋白酶活性(P)与凝乳活性(C)均较野生型低,但是其C/P值比野生型高1倍.     

胰岛素的体外折叠: 折叠中间体的鉴定 及其折叠途径

在单链胰岛素体外折叠研究的基础上, 研究了胰岛素的体外折叠过程. 在胰岛素的折叠过程中捕捉到6个主要的折叠中间体, 分别命名为P1A, P2B, P3A, P4B, P5B和P6B. 中间体的折叠实验证明6个中间体都能最终折叠成胰岛素. 在中间体的鉴定和分析以及推断的过渡态中间体形成和作用的基础上, 提出了胰岛素体外折叠的两步折叠途径. (ⅰ) 在折叠过程的早期, 完全还原的胰岛素, 即A链和B链各自形成中间体: 2个A链中间体(P1A和P3A), 4个B链中间体(P2B, P4B, P5B和P6B). (ⅱ) 在进一步的折叠过程中, 推断相继产生3个过渡态中间体: 首先, 中间体P3A通过分子内巯基/二硫键交换反应形成过渡态中间体Ⅰ, 该中间体含有1个天然二硫键A6-A11和2个巯基分别位于A7和A20; 然后, 过渡态中间体Ⅰ的巯基和P4B或P6B上的巯基通过氧化反应(主要以折叠体系中的GSSG为氧化剂)分别形成含有2个天然二硫键的过渡态中间体Ⅱ和Ⅲ; 最后, 过渡态中间体Ⅱ和Ⅲ通过分子内巯基/二硫键交换反应形成第3个天然二硫键, 完成胰岛素体外折叠.     

胰岛素的体外折叠: 折叠中间体的鉴定 及其折叠途径

在单链胰岛素体外折叠研究的基础上, 研究了胰岛素的体外折叠过程. 在胰岛素的折叠过程中捕捉到6个主要的折叠中间体, 分别命名为P1A, P2B, P3A, P4B, P5B和P6B. 中间体的折叠实验证明6个中间体都能最终折叠成胰岛素. 在中间体的鉴定和分析以及推断的过渡态中间体形成和作用的基础上, 提出了胰岛素体外折叠的两步折叠途径. (ⅰ) 在折叠过程的早期, 完全还原的胰岛素, 即A链和B链各自形成中间体: 2个A链中间体(P1A和P3A), 4个B链中间体(P2B, P4B, P5B和P6B). (ⅱ) 在进一步的折叠过程中, 推断相继产生3个过渡态中间体: 首先, 中间体P3A通过分子内巯基/二硫键交换反应形成过渡态中间体Ⅰ, 该中间体含有1个天然二硫键A6-A11和2个巯基分别位于A7和A20; 然后, 过渡态中间体Ⅰ的巯基和P4B或P6B上的巯基通过氧化反应(主要以折叠体系中的GSSG为氧化剂)分别形成含有2个天然二硫键的过渡态中间体Ⅱ和Ⅲ; 最后, 过渡态中间体Ⅱ和Ⅲ通过分子内巯基/二硫键交换反应形成第3个天然二硫键, 完成胰岛素体外折叠.     

人胰岛素突变体的分离纯化及性质研究

将人胰岛素原突变体(A4Glu→Leu)基因重组到pBV220表达载体上,在E.coli系统中得到高效表达,表达产物经SephadexG-50柱层析分离以及胰蛋白酶和羧肽酶B的酶促转化等步骤,可得到纯的人胰岛素突变体(A4Glu→Leu),其氨基酸组成与预期值相符,其受体结合活性及生物活性与标准猪胰岛素的基本相同.     

人胰岛素突变体GIRR的分离纯化及性质研究

构建人胰岛素原突变体GIRR(A2 1Asn→Gly,B1 7Leu→Ile ,B31Arg ,B32Arg)基因的原核融合表达载体并进行表达和纯化 ,进一步对其性质进行研究。将人胰岛素原突变体基因重组到pTXB1融合表达载体上 ,在E .coliBL2 1 (DE3)中得到高效表达。表达产物经几丁质亲和柱层析分离以及胰蛋白酶和羧肽酶B的酶促转化等步骤 ,得到纯的人胰岛素突变体GIRR ,其纯度经HPLC鉴定达到 95 % ,氨基酸组成和设计相符 ,其受体结合活性是猪胰岛素的 1 0 5 % ,生物活性为 2 6.8IU/mg。其血浆半衰期为 2 0分钟 ,比标准猪胰岛素提高 3倍多。结果提示本突变体具有更长的半衰期 ,为长效胰岛素的制备提供了新的思路。     

一种人胰岛素类似物的制备、鉴定与活性研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B25和B26位之间插入Glu,构建了［B10 Glu,B25-Glu-B26］胰岛素原融合蛋白基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性、凝胶过滤等步骤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经过DEAE离子交换和RP-HPLC纯化得到胰岛素突变体类似物,并经过质谱测定鉴定.凝胶过滤法测定了蛋白质分子自身的缔合性质,圆二色谱（CD）测定了构象的变化.并分别测定了放免活性、受体结合活性及小白鼠低血糖惊厥实验.结果表明,突变体分子缔合性明显下降.放免活性和受体结合活性分别约为标准胰岛素的63.5%和114.4%, 整体活力略高于天然胰岛素.     

Thermobifida fusca角质酶突变体D204C/E253C高效可溶表达及其应用

Thermobifida fusca角质酶突变体D204C/E253C是通过在野生型角质酶中引入二硫键获得的热稳定性突变体.由于二硫键的引入,导致突变体D204C/E253C在表达过程中,易错误折叠形成大量包涵体,可溶表达比率极低.这极大地限制了其发酵制备以及在PET纤维改性中的应用.为了加强突变体D204C/E253C的可溶表达,本研究将突变体D204C/E253C和周质蛋白二硫键氧化还原酶DsbC在大肠杆菌突变株Es-cherichia coli Origami B(DE3)中共表达,来异构化异常的二硫键.实验结果表明,共表达的角质酶突变体D204C/E253C正常折叠表达,酶活为61 U/mL,是非共表达的6.8倍;共表达的突变体D204C/E253C在80℃的半衰期可达16 h,能够在80℃对PET纤维进行改性.本研究通过与分子伴侣蛋白DsbC在大肠杆菌突变株E.coli Origami B(DE3)中的共表达,实现角质酶突变体D204C/E253C的高效可溶表达,为大规模工业应用提供了一定的理论基础.     

核苷二磷酸激酶A的定点突变及C4S突变体的制备和活性研究

目的:对核苷二磷酸激酶A(NDPK-A)二硫键异构的关键残基C4进行定点突变,构建、表达并纯化C4S突变体,测定其磷酸转移酶活性和DNase活性,研究二硫键异构对NDPK-A活性的影响。方法:以pBV220-nm23-H1质粒作为模板,通过设计合适的引物对NDPK-A进行定点突变,将第4位半胱氨酸突变为丝氨酸,构建NDPK-AC4S突变体;在大肠杆菌BL21中表达,DEAE-sepharose Fas tFlow与Cibacron Blue 3GA Sepharose CL-4B纯化目的蛋白,获得均一重组蛋白,纯度达到98%;DNA序列测定及重组蛋白的肽质量指纹图谱(PMF)分析均证明构建正确突变体;高效液相色谱法(HPLC)与DNA消化法分别测定野生型NDPK-A与C4S突变体的磷酸转移酶活性与DNase活性差异。结果:NDPK-AC4S突变体的磷酸基转移酶与DNase酶活性均高于野生型NDPK-A。结论:NDPK-A缺失二硫键后,活性增高。NDPK-A形成链内二硫键可能是其活性负调控模式之一。     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用

在前期研究中,已发现人瘦素（leptin）在体外再折叠过程中会形成稳定的二聚体,但其二聚化机制尚不清楚. 本研究旨在分析瘦素二聚体的结构特性,并重点研究体外再折叠过程中瘦素二聚化的机制. 相较与瘦素单体,瘦素二聚体保留了约75％免疫活性及15％受体结合活性,同时显示出明显慢的天然电泳迁移率. 圆二色性分析显示,二聚体基本保留了单体α螺旋索结构特征. 还原性及非还原性凝胶电泳分析和自由巯基测定结果表明,瘦素二聚体是由一对分子间二硫键连接2个单体而成的.为了确定瘦素二聚化过程中起主导作用的分子间二硫键,利用PCR定点突变技术构建了C96S和C146S两个突变体瘦素. 通过分析C96S及C146S突变体瘦素的体外再折叠特性及过程,并与野生型瘦素相比较,揭示C96S瘦素的二聚体显示出与野生型瘦素二聚体相似的特性,而C146S瘦素不能形成结构稳定的二聚体. 以上研究结果表明,Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用.     

Synthesis of 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone

The fermentation of dehydroepiandrosterone synthesized from the starting material diosgenin using Mucor racemosus produced 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone. The bioactivity of the microbial metabolites is also discussed. The species M. racemosus was isolated by screening among stains from soil samples collected from various parts of China.     

Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid

7-Fluoro sialic acid was prepared and activated as cytidine monophosphate (CMP) ester. The synthesis started with d-glucose, which was efficiently converted into N-acetyl-4-fluoro-4-deoxy-d-mannosamine. Aldolase catalyzed transformation yielded the corresponding fluorinated sialic acid which was activated as CMP ester using three different synthetases in the presence as well as in the absence of pyrophosphatase which possesses inhibitory properties. Finally, conditions were optimized to perform a one-pot reaction starting from fluorinated mannosamine, which yielded the 7-fluoro-7-deoxy-CMP-sialic acid by incubation with three enzymes.     

Karyotype instability with multiple 7/14 and 7/7 rearrangements

Summary Chromosomes were studied in a mentally retarded boy with microcephaly, growth retardation, facial erythema, café-au-lait spots, and IgA deficiency. In the lymphocytes there was a remarkable tendency to exchange parts of the chromosomes Nos. 7 and 14, the translocations almost exclusively taking place in bands 7p13, 7q32 and 14q11. Seven different types of rearrangements between Nos. 7 and 14, and some other chromosomal aberrations were found. No abnormalities could be detected in the bone marrow. The patient somewhat resembles those affected with ataxia-telangiectasia or with Bloom's syndrome, but on clinical and cytogenetic grounds these disorders could be excluded.7/14 Translocations similar to those found in our patient's lymphocytes have been reported to occur very rarely in the lymphocyte cultures of individuals with apparently normal chromosome constitution. A relationship between these phenomena may exist.     

Duplex Stability of Oligonucleotides Containing 7-Substituted 7-Deaza- and 8-Aza-7-Deazapurine Nucleosides

Abstract

The synthesis of 7-substituted 7-deaza- and 8-aza-7-deazapurine 2′-deoxyribonucleosides, their incorporation into oligonucleotides, and the stability of corresponding duplexes is described.     

Spinocerebellar ataxia 7 (SCA7)

Spinocerebellar ataxia 7 (SCA7) is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia associated with progressive macular dystrophy. The disease affects primarily the cerebellum and the retina, but also many other CNS structures as the disease progresses. SCA7 is caused by expansion of an unstable trinucleotide CAG repeat encoding a polyglutamine tract in the corresponding protein, ataxin-7. Normal SCA7 alleles contain 4-35 CAG repeats, whereas pathological alleles contain from 36-306 CAG repeats. SCA7 has a number of features in common with other diseases with polyglutamine expansions: (i) the appearance of clinical symptoms above a threshold number of CAG repeats (>35); (ii) a correlation between the size of the expansion and the rate of progression of the disease: the larger the repeat, the faster the progression; (iii) instability of the repeat sequence (approximately 12 CAG/transmission) that accounts for the marked anticipation of approximately 20 years/generation. The CAG repeat sequence is particularly unstable and de novo mutations can occur during paternal transmissions of intermediate size alleles (28-35 CAG repeats). This can explain the persistence of the disease in spite of the anticipation that should have resulted in its extinction.     

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.     

Immune Checkpoints of the B7 Family. Part 2. Representatives of the B7 Family B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, and ILDR2 and Their Receptors

Russian Journal of Bioorganic Chemistry - Immune checkpoints regulate polarity, strength, and termination of the immune response. The leading roles in these processes are played by molecules of the...     

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.     

Novel cytotoxic 7-iminomethyl and 7-aminomethyl derivatives of camptothecin

A series of new 7-iminomethyl derivatives of camptothecin were obtained from camptothecin-7-aldehyde and aromatic, alicyclic and aliphatic amines. Their hydrogenation led to the corresponding amines. All the imines and the less polar amines showed a marked increase of the cytotoxic activity against H460 non-small lung carcinoma cell line, with respect to topotecan. The lipophilicity of the substituent in position 7 of camptothecin seems to play an important role for cytotoxic potency. The 7-phenyliminomethyl derivative showed efficacy comparable to topotecan in vivo against NSCLC H460 xenografted in athymic nude mice.