江浙蝮蛇毒碱性磷脂酶A2的溶血位点

用聚合酶链式反应 (PCR)对江浙蝮蛇毒碱性磷脂酶A2 基因进行点突变 ,使对应 34位氨基酸残基由Arg分别突变为Glu和Gln ,将其基因克隆至温敏表达载体pBLMVL2 ,并在大肠杆菌RR1中成功诱导表达 ,表达产物以包涵体的形式存在。对包涵体进行变复性后 ,用凝胶过滤的方法纯化。对纯化后的产物进行酶活力测定及溶血活性测定。实验结果表明 ,突变后的表达产物维持原有磷脂酶A2 活性 ,且溶血活性显著降低 ,表明磷脂酶A2 的34位碱性氨基酸残基在溶血过程具有重要作用     

大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析

为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH, d-3-phosphoglycerate dehydrogenase, EC 1.1.1.95）,通过对其碱基序列和蛋白质结构分析,用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407~410位氨基酸）、M3（缺失337~410位氨基酸）。M0(野生型)及各突变型基因与pET22b(+)载体连接后,表达融合蛋白。在非变性条件下,由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白。酶活性测定结果表明,M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性,且部分解除了终产物L-丝氨酸的反馈抑制作用;M3蛋白酶完全解除了终产物的反馈抑制作用,但酶本身的催化活性略有降低（为野生型的83%）。M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7 μmol/L、20 μmol/L、50 μmol/L,说明该酶C-末端1~4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响。     

枯草杆菌谷氨酰胺转胺酶的克隆及其在大肠杆菌中的融合表达

利用PCR技术 ,从枯草杆菌DB40 3染色体上扩增出谷氨酰胺转胺酶基因 ,将其克隆到大肠杆菌载体pET32a( + )中 ,成功构建谷氨酰胺转胺酶表达载体pET32-BTGase ,并转化大肠杆菌BL2 1 (DE3)。重组克隆在IPTG诱导下 ,表达出硫氧还蛋白 谷氨酰胺转胺酶 (Trx-BTGase)融合蛋白 ,表达量占细菌总蛋白量的 2 6%。利用金属螯合层析纯化菌体裂解上清中表达的融合蛋白 ,纯度超过 80 %,再通过分子筛层析进一步纯化得到融合蛋白纯品。酶活性分析表明表达的Trx-BTGase融合蛋白具有交联蛋白的活性 ,并发现Trx-BTGase融合蛋白和经凝血酶酶切后得到的BTGase单体都能催化牛血清白蛋白的聚合反应     

人分泌型磷脂酶A2 GIIE的E54突变及其对底物选择性的影响

人分泌型磷脂酶A2 GIIE (Human secreted phospholipase A2 GIIE,hGIIE) 通过发挥酶催化作用,参与炎症反应和脂代谢过程。为了揭示hGIIE的底物选择机制,文中对hGIIE进行了定点突变,采用毕赤酵母Pichia pastoris重组表达突变体蛋白,然后通过阳离子交换和分子排阻两步法纯化蛋白,最后用等温微量热滴定仪测定酶活性。hGIIE的结构分析显示,氨基酸E54可能与GIIE的底物头基选择性有关,经过同源序列比对,拟将E54突变为丙氨酸 (A)、苯丙氨酸 (F) 和赖氨酸 (K)。突变体E54A、E54F和E54K在毕赤酵母组成型表达系统中实现重组表达,通过两步纯化,纯度达到90%以上。酶活性实验显示,突变体与底物1,2-二己酰卵磷脂 (1,2-dihexanoyl-sn- glycero-3-phosphocholine,DHPC) 和1,2-二己酰磷酸甘油 (1,2-dihexyl phosphate glycerol,DHPG) 的亲和力发生改变,其中突变体E54K与DHPG的Km值为突变前的0.39倍,亲和力明显增强;突变体E54F与DHPC的Km值为突变前的1.93倍,亲和力明显减弱。hGIIE的E54突变体蛋白与磷脂底物的亲和力发生明显改变,说明E54在hGIIE的磷脂底物选择性水解过程中起重要作用。     

人巨细胞病毒M抗原表位保守氨基酸突变的分析

为确定人巨细胞病毒M抗原表位MAD的关键氨基酸残基, 以MAD多肽序列为基础, 分别将保守氨基酸残基单一突变为甘氨酸残基, 构建各自突变体, 然后与人源Fc的N端融合, 通过原核表达载体pET32-Fc表达融合蛋白MAD-Fc, 经protein A柱亲和纯化得到各突变体纯品。通过ELISA及Western blotting方法验证各突变体特异结合羊抗HCMV多抗间的差异, 从而确定表位关键氨基酸残基。结果显示, 将MAD中的谷氨酰胺残基单突变为甘氨酸残基后, MADQ-G结合羊抗HCMV多抗活性大大降低, 差异显著; 而其他氨基酸残基单突变时, 对MAD活性影响程度很小。由此得出结论: MAD结合羊抗HCMV多抗的活性与谷氨酰胺残基有关。     

人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。     

人类免疫缺陷病毒1型Tat蛋白N末端缺失突变体融合蛋白的表达及其免疫原性分析

本研究采用PCR方法从人类免疫缺陷病毒1型（Human immunodeficiency virus 1,HIV-1）HXB2株tat基因中扩增编码Tat蛋白N末端1-21位氨基酸缺失的突变体Tat22-101基因片段,构建其原核表达质粒pET32a-Tat22-101,经双酶切及测序验证后,转化大肠埃希菌BL21（DE3）,进行IPTG诱导表达及Ni²⁺-NTA柱亲和层析纯化。纯化后的突变体融合蛋白PET32a-Tat22-101经SDS-PAGE及Western blotting鉴定,其相对分子质量约为26.9kD。该融合蛋白免疫BALB/c小鼠,经ELISA检测结果表明,pET32a-Tat22-101融合蛋白不仅较好地保留其免疫原性,而且能诱导产生高滴度的针对Tat N末端区之外的Tat其他功能区表位的抗体,为进一步研究Tat生物学功能和研制新型HIV Tat疫苗奠定试验基础。     

SARS冠状病毒蛋白酶3CLpro N-finger多肽是酶活性所必需

构建了SARS冠状病毒主要蛋白酶3CL^pro及其缺失N端七肽即N-finger的突变体3CL^pro（△1-7）融合表达质粒,并于大肠杆菌表达系统中表达。得到的融合蛋白经肠激酶酶切,亲和层析,最终得到纯化的3CL^pro。及其突变体3CL-（△1-7）。酶活性实验显示,去除N-finger多肽的突变体3CL^pro（△1-7）丧失了3CL^pro所具有的对含有其自动水解位点的荧光底物的水解活性,表明处于非酶活性中心的N—finger多肽是酶活性所必需的。利用固相合成法,进一步合成了N—finger七肽。酶抑制实验表明N—finger七肽表现了对3CL^pro部分竞争抑制活性。     

大肠杆菌CM/PDT抗反馈抑制突变体的构建及其性质

为了获得具有高催化活性且抗反馈抑制的大肠杆菌分支酸变位酶 预苯酸脱水酶 (chorismatemutase prephenatedehydrataseCM PDT) [EC5 .4 .99.5 EC4 .2 .1.5 1],通过相关菌种CM PDT氨基酸序列同源比较 ,寻找高度保守位点 .用定点突变及PCR法构建突变酶M1(缺失 30 4T、30 5G、Q30 6K)、M2 (缺失W 338)、M3(缺失 30 1～ 386位氨基酸 )、M32 9(E32 9A)和M374 (C374A) ,野生型及各突变型基因与pET2 8a(+ )载体连接后 ,表达融合蛋白 .在非变性条件下 ,由TALON金属螯合亲和层析柱纯化野生型和突变体的酶蛋白 .酶活性测定表明 ,突变体M3的PDT活性下降为野生型活性的 2 9% ,但保持了CM活性 .突变体M374保持了CM ,PDT两种酶的活性 ,突变体M1、M2、M32 9的CM ,PDT活性有一定程度的提高 .酶抗反馈抑制作用检测表明 ,突变体M3、M374解除了苯丙氨酸的反馈抑制作用 ,M1、M2、M32 9部分解除了苯丙氨酸的反馈抑制作用 .与含野生型pheA基因的E .coliBL2 1菌株相比 ,含突变基因的E .coliBL2 1菌株对 10mmol L的苯丙氨酸代谢类似物具有强的抗反馈抑制作用 ,其中M1,M2 ,M3对 2 0mmol L的类似物具有抗反馈抑制作用     

脆性组氨酸三联体与复制蛋白A相互作用关键氨基酸残基的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR/CHK1通路的影响,在确定Fhit与复制蛋白A（RPA）存在相互作用的基础上鉴定Fhit与RPA相互作用的关键氨基酸残基,为进一步研究Fhit特异的信号通路奠定基础。方法：构建一系列Fhit缺失突变体基因Fhit1～Fhit11及6种Fhit点突变体基因,将这些基因插入含GST基因的原核表达载体中,在大肠杆菌中表达并纯化GST-Fhit1～GST-Fhit11融合蛋白、突变体GST-FhitSIYEEL、GST-FhitIY、GST-FhitEL、GST-FhitF、GST-FhitA,以及GST-FhitD融合蛋白,用GST沉降技术研究Fhit与RPA相互作用的关键氨基酸残基。结果：Fhit蛋白第112～117（SIYEEL）残基可能是Fhit与RPA相互作用的关键区域,而第114（Y）残基可能是Fhit与RPA相互作用的关键氨基酸残基。结论：确定了Fhit与RPA相互作用的关键氨基酸残基,为阐明Fhit在维持基因组完整性方面的机理提供了线索。     

Purification, characterization and gene cloning of a novel phospholipase A2 from the venom of Agkistrodon blomhoffii ussurensis

A new phospholipase A2 with Gln at the site 49, abbreviated as Gln49-PLA2, has been purified from the venom of Agkistrodon blomhoffii ussurensis by using ion-exchange chromatography, gel filtration chromatography and reversed-phase HPLC, and behaves as a single-band on SDS-PAGE. Its molecular weight is 13881.85+/-0.33 Da given by mass spectrometry and pI is about 8.56 given by isoelectric focusing. Gln49-PLA2 does not show phospholipase A2 and hemorrhagic activity, whereas shows weak toxic and apparent anticoagulant activity. Based on the N-terminal sequencing and peptide mass fingerprint analysis, Gln49-PLA2 cDNA has been cloned by means of RT-PCR. Gln49-PLA2 consists of 122 amino acid residues and has the structural features of class II of snake venom phospholipase A2.     

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A₂ homologue: insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.     

A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity

The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.     

Factor XIIIa mediated attachment of S. aureus fibronectin-binding protein A (FnbA) to fibrin: identification of Gln103 as a major cross-linking site

In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A2 (A .aBPLA2 )基因克隆至温敏表达载体 pBLMVL2 ,在大肠杆菌RR1中成功诱导表达 .表达产物A .aBPLA2 约占细菌蛋白质总量的 2 0 % ,并以包涵体的形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用FPLCSuperoseTM12纯化 ,产物经过SDS 聚丙烯酰胺凝胶电泳检测只有单一条带 .对纯化后的表达A .aBPLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,表达A .aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2 酶活性相近 ,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2 的溶血活性 ,没有抑制血小板聚集活性 .最后对磷脂酶A2 的结构与这些活性的关系进行了讨论     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ , 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化, 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论     

New beta -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 and its roles on the morphological differentiation

A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta-lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta-lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.     

尖吻蝮蛇毒酸性磷脂酶A_2Ⅱ的表达及其生化特征

将尖吻蝮蛇毒酸性磷酯酶 A2 ( A.a APLA2 )基因克隆至温敏表达载体 p BLMVL2 ,在大肠杆菌 RR1中成功诱导表达 .表达产物 A.a APLA2 约占细菌蛋白质总量 2 5 % ,并以包涵体形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用 FPLC Superose TM1 2纯化 ,产物经过 SDS- PAGE检测只有单一条带 .对纯化后的表达 A.a APLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,A.a APLA2 的酶活性处于变性后复性江浙蝮蛇酸性磷脂酶 A2 和碱性磷脂酶A2 的酶活性之间 ,没有抑制血小板聚集活性 ,只有微弱溶血活性 .最后对 PLA2 的结构与这些活性的关系进行了讨论