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Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

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Acta Biochimica et Biophysica Sinica 2005年第11期37卷 737-742页

作者： Jin-Peng GAO Zhen-Hua YONG Feng ZHANG Kang-Cheng RUAN Chun-He XU Gen-Yun CHEN institute of Plant Physiology & Ecology.shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china laboratory of proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china College of sciences,Zhejiang University.Hangzhou 310029,china Graduate School of the chinese academy of sciences,shanghai 200032,china institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China Graduate School of the chinese academy of sciencesshanghai 200032 China College of sciences Zhejiang UniversityHangzhou 310029 China laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

To determine the contribution of charged amino acids to binding with the photosystem Ⅱcomplex(PSⅡ),the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate(NSP)or glycine methyl ester(GME)in the presence of a water-soluble carbodiimide,*** on isoelectric point shift,4-10 and 10-14 amino groups were modified in the presence of2 and 4 mM NSP,***,3-4 carboxyl groups were modified by reaction with *** of negatively charged carboxyl groups with GME did not alter the binding activity of theextrinsic 18 kDa ***,the NSP-modified 18 kDa protein,in which the positively charged aminogroups had been modified to uncharged methyl esters,failed to bind with the PSⅡ membrane in the presenceof the extrinsic 23 kDa *** defect can not be attributed to structural or conformational alterationsimposed by chemical modification,as the fluorescence and circular dichroism spectra among native,GME-and NSP-modified extrinsic 18 kDa proteins were ***,we have concluded that the positive chargesof lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSⅡ membranes in thepresence of the extrinsic 23 kDa ***,it was found that the negative charges of carboxylgroups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSⅡmembranes.

关键词： extrinsic 18 kDa protein circular dichroism electrostatic interaction chemical modification

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Expression of Overlapping PreS1 Fragment Recombinant Proteins for the Determination of Immunogenic Domains in HBsAg PreS1 Region

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 397-404页

作者： Wei-Guo HU, Jun WEI&, Xin-Xiu YANG, Heng-Chuan XIA, Feng LI, and Zu-Chuan ZHANG* Key laboratory of proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences,the chinese academy of sciences, shanghai 200031, china Key laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of SciencesShanghai 200031 China

In this study, eight preS1 fragments overlapped in preS1 (21–119) region of HBV adr subtype, i.e. preS1 (21–47), preS1 (34–59), preS1 (48–70), preS1 (60–85), preS1 (71–94), preS1 (86–109), preS1 (95–119) and preS1 (21–119), were cloned by PCR, and expressed as GST fusion proteins. These GST- preS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21– 119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21–59) in N-terminus and preS1 (95–109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34–59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21–119) region. The results here would be helpful for the design of new vaccines against HBV.

关键词： HBV preS1 overlapping fragment immunogenic domain

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Preparation and Preliminary Application of Monoclonal Antibodies against Trichokirin-S1,a Small Ribosome-inactivating Peptide from the Seeds of Trichosanthes kirilowii

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 447-452页

作者： Xin-Xiu YANG Feng LI Wei-Guo HU Heng-Chuan XIA Zu-Chuan ZHANG Key laboratory of proteomics.institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Key laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Trichokirin-S1,a small ribosome-inactivating peptide recently purified from the seeds ofTrichosanthes kirilowii,has potential clinical applications because of its small molecular *** stablestrains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) againstTrichokirin-S1 have been developed using the hybridoma *** isotypes of these two mAbs,1F11and 2A5,were determined to be IgGand IgG,*** affinity constants,which were measuredby non-competitive ELISA,were found to be 2.3×10~8 Mand 2.8×10~8 M,*** immunoaffinitymethod using 2A5-coupled Sepharose 4B was successfully developed to purify *** twoantibodies have also been used to detect Trichokirin-S1 in Western blot.

关键词： Trichokirin-S1 monoclonal antibody Western blot affinity chromatography

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Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 (HVMLC1) for Functional Studies

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Acta Biochimica et Biophysica Sinica 2006年第9期38卷 625-632页

作者： Zhen-Yan FU~1 Bao-Tong XIE~2 Yi-Tong MA~1* Zu-Xun GONG~2* 1 Cardiovascular Research Center,First Clinical College of Xinjiang Medical University,Urumqi 830054,china 2 Key laboratory of proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Cardiovascular Research Center First Clinical College of Xinjiang Medical UniversityUrumqi 830054 China Key laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

Using purified recombinant human ventricular myosin light chain 1 （HVMLC 1） as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were *** experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or *** was also demonstrated that C8 was directed against the NN part of theN-fragment （amino acid 1-40） of HVMLC1,and both C9 and B12 against the C-fragment （amino acid 99-195）.The affinity constants of C8,C9 and B12 were 3.20×10～8,8.60×10～7 and 1.77×10～8 M-1,respectively,determined by non-competitive *** isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were *** the presence of C9 or B12,the actin-activated Mg2+ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg2+ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin （myosin B） and reconstituted actomyosin,but C8 did *** results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of *** inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg2+ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.

关键词： human ventricular myosin light chain 1 (HVMLC1) monoclonal antibody Mg2+ATPase activity superprecipitation

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Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

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cell Research 2003年第5期13卷 369-374页

作者： HENG CHUAN XIA, FENG LI, ZHEN LI, Zu CHUAN ZHANG~* Key laboratory of proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 200031, china. State Key laboratory of Reproductive biology Institute of Zoology Chinese Academy of SciencesBeijing 100080 China Department of Veterinary Pathobiology University of Missouri-Columbia MO 65211 USA

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin （Cucurbita moschata） has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of～29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

关键词： ribosome-inactivating protein (RIP) Moschatin immunotoxin.

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Six Specific Lysihe Residues are Crucial in Maintaining the Structure and Function of Soluble Manganese Stabilizing Protein

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Acta Biochimica et Biophysica Sinica 2006年第9期 611-619页

作者： Jin-Peng GAO~(1,2#) Feng ZHANG~(1,3#) Li ZHANG~4 Yin-Long GUO~4 Kang-Cheng RUAN~5 De-An JIANG~3* Chun-He XU~1 1 institute of Plant Physiology and Ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china 2 Graduate School of the chinese academy of sciences,shanghai 200032,china 3 College of Life sciences,Zhejiang University,Hangzhou 310029,china 4 shanghai Mass Spectrometry Center,shanghai institute of Organic Chemistry,chinese academy of sciences,shanghai 200031,china 5 laboratory of proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china

When manganese stabilizing protein （MSP） was treated with 0.5mM N-succinimidyl propionate（NSP）,the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered,in spiteof changes of MSP surface Lys ***,far-ultraviolet circular dichroism and intrinsicfluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondaryand tertiary *** of the sites of NSP modification by Staphylococcus V8protease digestion ofthe modified protein,as well as analysis by matrix-assisted laser desorption ionization-time of flight massspectrometry,indicated that seven Lys residues were *** results suggested that these residues arenot absolutely essential to the structure and function of ***,when the NSP concentration wasincreased to 4mM,the modified MSP was unable to bind photosystem Ⅱ and completely lost its *** far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clearconformational change in MSP after 4 mM NSP treatment,suggesting that some Lys residues are involved inmaintaining the structure and function of *** by matrix-assisted laser desorption ionization-timeof flight mass spectrometry indicated that another six Lys residues,namely Lys20,Lys101,Lys196,Lys207,Lys130 （or Lys137） and Lys66 （or Lys76）,were modified by 4 mM ***,these six Lys residuesare crucial in maintaining the structure and function of soluble MSP.

关键词： chemical modification fluorescence spectrum circular dichroism spectrum manganese stabilizing protein Lys residue

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锤头状核酶在鸡胚成纤维细胞中的对鹅副粘病毒SF02繁殖的抑制作用(英文)

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生物化学与生物物理学报 2003年第9期35卷 801-805页

作者：邹键龚祖埙中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学重点实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学重点实验室上海200031

针对鹅副粘病毒SF0 2的F基因 ,设计了锤头状核酶RzF5 98,并插入真核细胞表达载体pcDNA3获得该核酶的转基因质粒pcDNA RzF5 98。以无核酶功能的突变体dRzF5 98和pcDNA3空质粒为对照 ,分析核酶对病毒繁殖的抑制作用。结果表明 ,RzF5 98不... 详细信息

针对鹅副粘病毒SF0 2的F基因 ,设计了锤头状核酶RzF5 98,并插入真核细胞表达载体pcDNA3获得该核酶的转基因质粒pcDNA RzF5 98。以无核酶功能的突变体dRzF5 98和pcDNA3空质粒为对照 ,分析核酶对病毒繁殖的抑制作用。结果表明 ,RzF5 98不仅能在体外成功切割靶基因的mRNA ,还能有效抑制SF0 2在鸡胚成纤维细胞中的繁殖。转染pcDNA RzF5 98的鸡胚成纤维细胞经SF0 2感染后存活率可达 80 %左右 ,而未转染任何质粒的细胞和转染pcDNA3空质粒的细胞经SF0 2感染后存活率只有约 5 %。转染dRzF5 98的细胞对病毒也具有一定抗性 ,这可能是由于反义RNA的作用。

关键词：锤头状核酶鹅副粘病毒新城疫病毒鸡胚成纤维细胞

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慈菇蛋白酶抑制剂A和B的活性中心探讨(英文)

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生物化学与生物物理学报 2002年第5期34卷 662-666页

作者：李炯阮康成戚正武中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学实验室上海200031

慈菇蛋白酶抑制剂A和B(APIA和APIB)是一种双头多功能抑制剂 ,它们的一级结构和cDNA序列已经被阐明。为了找到它们的活性中心 ,利用定点诱变的方法将APIB中根据与其他抑制剂家族的序列比较所推断的可能的活性中心残基 :Lys44、Arg76和Ar... 详细信息

慈菇蛋白酶抑制剂A和B(APIA和APIB)是一种双头多功能抑制剂 ,它们的一级结构和cDNA序列已经被阐明。为了找到它们的活性中心 ,利用定点诱变的方法将APIB中根据与其他抑制剂家族的序列比较所推断的可能的活性中心残基 :Lys44、Arg76和Arg87分别用Pro替代 ,所得到的突变基因分别在酵母分泌体系中得到了表达。与天然的APIB相比 ,K44P APIB对胰蛋白酶的抑制活力没有改变 ;而R76P APIB和R87P APIB对胰蛋白酶的抑制活力都分别下降了一半 ,由原来的抑制两分子变成了一分子。表明Arg76和Arg87分别是APIB的两个活性中心残基 ,而Lys44则不是。为了证实以上结论 ,进一步制备了另外 3种突变体 (K44P R76P APIB、K44P R87P APIB、R76P R87P APIB) ,在每个突变体中 ,3个可能的活性位点中只保留 1个。有关的抑制活力测定表明 ,K44P R76P APIB(只保留Arg87)和K44P R87P APIB(只保留Arg76)分别只抑制一分子胰蛋白酶 ,而R76P R87P APIB(只保留Lys44)对胰蛋白酶基本不抑制 ,从而肯定了以上结论。经过测定 ,两个突变体K44P R87P APIB和K44P R76P APIB对胰蛋白酶的抑制常数Ki 分别是 0 .39nmol/L和 0 .4 7nmol/L。突变体R87L APIB(APIA中 87位是Leu)丧失了接近一半的胰蛋白酶抑制活力 ,但同时对胰凝乳蛋白酶的抑制活性由原来?

关键词：慈菇蛋白酶抑制剂定点诱变抑制活性活性中心

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一种黑豆蚜储存蛋白质的分离纯化及其亚基的性质

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生物化学与生物物理学报 2003年第2期35卷 189-192页

作者：甘明丁德诚周元聪中国科学院上海生命科学研究院植物生理生态研究所中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学重点实验室上海200032 上海200031

从黑豆蚜 (AphiscraccivoraKoch)血淋巴中经DE5 2纤维素离子交换层析、SephadexG 15 0凝胶过滤以及在FPLC上QSepharose分离出一种蛋白质。该蛋白质经SDS PAGE测定在非还原和还原条件下分子量均为 6 0kD左右 ,等电聚焦电泳测得其等电点约... 详细信息

从黑豆蚜 (AphiscraccivoraKoch)血淋巴中经DE5 2纤维素离子交换层析、SephadexG 15 0凝胶过滤以及在FPLC上QSepharose分离出一种蛋白质。该蛋白质经SDS PAGE测定在非还原和还原条件下分子量均为 6 0kD左右 ,等电聚焦电泳测得其等电点约为 5 .0 ,为糖蛋白。若蚜期间该蛋白质在蚜虫体内积累 ,羽化至成蚜时含量明显减少 ,为蚜虫提供发育所需氨基酸 ,在成蚜期间仍以低浓度存在。根据其分子量和氨基酸组成与含量变化动态应属不完全变态昆虫的一种持续性储存蛋白质。

关键词：黑豆蚜储存蛋白质分离纯化

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水稻齿叶矮缩病毒S8基因编码的结构蛋白质及其自聚集性质

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生物化学与生物物理学报 2002年第5期34卷 565-570页

作者：吕慧涓邵朝纲龚祖埙中国科学院上海生命科学研究院生物化学与细胞生物学研究所、蛋白质组学重点实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所、蛋白质组学重点实验室上海200031

水稻齿叶矮缩病毒是一种以水稻为侵害寄主的植物呼肠孤病毒 ,其基因组 1 0条dsRNA编码的蛋白质产物的功能除S9外的大部分还未阐明。报道了该病毒菲律宾分离株S8的开放阅读框序列 ,在大肠杆菌中表达和纯化出了其 6 7kD的蛋白质产物 ,并... 详细信息

水稻齿叶矮缩病毒是一种以水稻为侵害寄主的植物呼肠孤病毒 ,其基因组 1 0条dsRNA编码的蛋白质产物的功能除S9外的大部分还未阐明。报道了该病毒菲律宾分离株S8的开放阅读框序列 ,在大肠杆菌中表达和纯化出了其 6 7kD的蛋白质产物 ,并进一步研究了该产物的功能。实验结果表明 ,S8的蛋白质产物是病毒的一种主要结构蛋白质 ,在体外具有自剪切活性和自聚集性质 ,并推测可能是病毒的内层衣壳蛋白

关键词： RRSV P8 结构蛋白质自剪切自聚集

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