葡萄球菌核酸酶R型的晶体生长及初步晶体学分析

葡萄球菌核酸酶Ｒ型是葡萄球菌核酸酶的一个类似物,已在大肠杆菌中高效表达并且用阳离子交换色谱纯化。应用悬滴气相扩散法在含５０％聚乙二醇的５０ｍｍｏｌ／Ｌ柠檬酸缓冲体系,ＰＨ５．６中获得适合Ｘ射线分析用的单晶体。     

人热休克蛋白转录因子1 DNA结合结构域的表达、纯化和晶体生长

目的:获得大量热休克转录因子1(HSF1)DNA结合结构域(DBD)蛋白,用于晶体生长的三维结构解析。方法:将DBD基因片段克隆至原核表达载体pGEX-6P-1中并获得高效表达,经过Glutathione SepharoseTM 4B亲和层析、ResourceQ纯化后,蛋白纯度达到95%以上。结果:圆二色谱仪分析蛋白质的二级结构结果显示α螺旋占33%,β折叠占15%;采用悬滴气相扩散法得到了针状DBD晶体。结论:纯化的蛋白质与同源性达68%的Kluyveromyceslactis的DBD有相似的空间构象。获得的蛋白质晶体为进一步的三维结构解析奠定了基础。     

重组人可溶性BAFF蛋白的原核表达、纯化及鉴定

目的:在大肠杆菌中表达人B细胞活化因子可溶性胞外域134~285(rhsBAFF134-285)蛋白,并进行纯化与鉴定。方法:重组表达质粒pET-32ammrhsBAFF在大肠杆菌Rosetta-gami B(DE3)中于16℃下进行IPTG诱导表达。经His亲和层析纯化得到融合蛋白后进行TEV蛋白酶酶切切除Trx融合标签,进而纯化得到rhsBAFF目的蛋白,SDS-PAGE、Western Blot和ELISA鉴定纯化产物。结果:Trx-rhsBAFF融合蛋白相对分子量约31000,16℃表达时主要以包涵体形式表达,上清中也有部分表达。融合蛋白纯化后经酶切再纯化得到相对分子量约17000、纯度95%以上的rhsBAFF目的蛋白,鉴定可被小鼠抗rhBAFF单克隆抗体和小鼠抗rhBAFF多抗血清特异性识别。结论:成功原核表达并纯化得到rhsBAFF蛋白,为进一步开发用于研究人类自身免疫性疾病的BAFF检测试剂盒奠定基础。     

6-羟基-3-琥珀酰吡啶单加氧酶的纯化与结晶条件

【目的】在大肠杆菌中克隆表达尼古丁降解关键的6-羟基-3-琥珀酰吡啶单加氧酶基因hspB,纯化重组HspB蛋白并进行结晶条件的初步研究。【方法】从恶臭假单胞菌S16基因组中PCR扩增hspB基因,构建重组表达载体pET28a-hspB,并在E.coli BL21(DE3)中诱导表达,利用亲和层析和凝胶过滤层析纯化重组蛋白。利用悬滴扩散法对HspB蛋白进行结晶条件筛选和优化。【结果】本文成功构建重组质粒pET28a-hspB并纯化获得达到结晶纯度的HspB蛋白。结晶条件初筛和优化后获得可培养HspB蛋白晶体的条件为0.2 mol/L NaCl、0.1 mol/L HEPES pH 7.5、1.1 mol/L(NH4)2SO4、4°C、加晶种。【结论】HspB蛋白纯化体系的构建和结晶条件的初步研究为从结构生物学的角度进一步研究HspB结构与功能的关系、定向进化提高HspB催化效率奠定了基础。     

结核分枝杆菌Rv3369基因的表达和纯化

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。     

限制性内切酶Mlu I蛋白及其硒代衍生物的制备

【背景】限制性内切酶Mlu I是一种常用的工具酶,在分子生物学领域发挥着重要的作用,其三维结构尚未被解析。【目的】在大肠杆菌中克隆表达、纯化重组Mlu I蛋白及其硒代蛋白,并进行结晶条件的研究。【方法】构建重组表达载体pET28b-Mlu I,在大肠杆菌BL21(DE3)pLysS中诱导表达,利用亲和层析和凝胶过滤层析纯化重组Mlu I蛋白和硒代Mlu I蛋白。对蛋白进行质谱检测、圆二色谱检测以及酶活检测,利用坐滴法进行结晶条件的筛选。【结果】构建了重组表达载体pET28b-Mlu I并纯化获得达到结晶纯度的蛋白,通过质谱检测确定硒代Mlu I蛋白中的8个甲硫氨酸全部被取代,结合酶活测试及圆二色谱检测确定了硒代对Mlu I蛋白的活性、结构无明显影响。采用坐滴法进行初步的晶体生长研究,重组蛋白目前已在1种条件下获得针状晶体并进行初步衍射,获得分辨率在0.32 nm左右的衍射数据。【结论】Mlu I蛋白及硒代Mlu I蛋白纯化体系的构建和结晶条件的研究,可为下一步解析Mlu I三维结构、作用机制的探讨及定向改造奠定基础。     

大肠杆菌肽酶D的重组表达和晶体生长

用PCR技术扩增大肠杆菌肽酶D(pepD)的基因,并将扩增基因克隆于原核表达载体pET-22b中,获得该蛋白的可溶性高效表达。通过镍亲和层析柱一步纯化后可得电泳纯的重组蛋白。该蛋白为二体形式,每个单体含有一个锌原子。用“悬滴气相扩散法”对该蛋白进行晶体生长,获得单晶。     

限制性内切酶Mlu Ⅰ蛋白及其硒代衍生物的制备

【背景】限制性内切酶Mlu Ⅰ是一种常用的工具酶,在分子生物学领域发挥着重要的作用,其三维结构尚未被解析。【目的】在大肠杆菌中克隆表达、纯化重组Mlu Ⅰ蛋白及其硒代蛋白,并进行结晶条件的研究。【方法】构建重组表达载体pET28b-Mlu Ⅰ,在大肠杆菌BL21(DE3)pLysS中诱导表达,利用亲和层析和凝胶过滤层析纯化重组Mlu Ⅰ蛋白和硒代Mlu Ⅰ蛋白。对蛋白进行质谱检测、圆二色谱检测以及酶活检测,利用坐滴法进行结晶条件的筛选。【结果】构建了重组表达载体pET28b-Mlu Ⅰ并纯化获得达到结晶纯度的蛋白,通过质谱检测确定硒代Mlu Ⅰ蛋白中的8个甲硫氨酸全部被取代,结合酶活测试及圆二色谱检测确定了硒代对Mlu Ⅰ蛋白的活性、结构无明显影响。采用坐滴法进行初步的晶体生长研究,重组蛋白目前已在1种条件下获得针状晶体并进行初步衍射,获得分辨率在0.32 nm左右的衍射数据。【结论】Mlu Ⅰ蛋白及硒代Mlu Ⅰ蛋白纯化体系的构建和结晶条件的研究,可为下一步解析Mlu Ⅰ三维结构、作用机制的探讨及定向改造奠定基础。     

人源Fank1蛋白质FN3结构域多肽的原核表达，纯化及晶体筛选

目的纯化人源Fank1（fibronectin type Ⅲ and ankyrin repeat domain1）蛋白质N端FN3（fibronectin typeⅢ,Ⅲ型纤黏连蛋白）结构域蛋白,用于晶体生长的三维结构分析。方法将FN3结构域基因片段克隆至原核表达载体pGEX-6P-1中,将菌落PCR和测序鉴定正确的重组质粒转化E.coli BL21（DE3）后获得表达菌株。该菌株经IPTG诱导高效表达出带有GST标签的可溶性的融合蛋白,经过Glutathione Sepha-rose^TM 4B亲和层析、Hiload16／60 superdex200分子筛层析纯化后,蛋白纯度达到95％以上。结果纯化蛋白采用悬滴气相扩散法得到棒状晶体。结论成功制备了高纯度FN3蛋白,获得FN3蛋白质晶体,为进一步的三维结构解析及Fank1功能研究奠定了基础。     

酿酒酵母RAVE复合物的155kD亚基Rav1p的克隆及其在大肠杆菌中的表达纯化

以酿酒酵母基因组DNA为模板,根据CenBank上公布的酿酒酵母Ravlp基因(rav1)序列和表达裁体特性设计 特异性引物,PCR扩增得到4 074 bp的DNA片段,将PCR产物和原核表达栽体pET28a(+)同时进行双酶切;双酶切后的PCR产物和表达栽体进行连接,构建成重组质粒pET28a-ravl.再将pET28a-ravl转化到BL21( DE3)感受态细胞中.经IPTG 16℃低温诱导40h表达His-tag融合的Ravlp.诱导后的菌体进行超声波破碎,然后用GE healthcare公司的AKTA蛋白纯化仪和His Trap HP I mL亲和层析柱纯化目的蛋白.SDS-PAGE电泳分析和Western blot分析显示在155 kD有明显的条带,成功实现了Ravlp在大肠杆菌中的表达纯化.     

Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester.

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given.     

Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester.

The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well.     

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.     

Synthesis of an ether-linked alkyl 5a-carba-beta-D-glucoside,a 5a-carba-beta-D-galactoside,a 2-acetamido-2-deoxy-5a-carba-beta-D-glucoside,and an alkyl 5a'-carba-beta-lactoside

For the purpose of providing biologically stable building blocks for the biocombinatorial synthesis using a living cell, some ether-linked alkyl 5a-carba-beta-D-glycoside primers were prepared. The key step of the synthesis was coupling of 1-bromo-n-alkanes with the 1-OH unprotected derivatives of 5a-carba-sugar analogues of D-glucose, D-galactose, and 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), in DMF in the presence of sodium hydride. Alternatively, alkyl carba-lactoside was synthesized by incorporation of a 5a-carba-beta-D-galactose residue into the 4-position of dodecyl beta-D-glucopyranoside. A strong and specific inhibition of beta-galactosidase (K(i) 0.67 microM, bovine liver) was found for dodecyl 5a-carba-beta-D-galactopyranoside.     

Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships

Background

Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.

Results

Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R² = 0.88) and rat tissues (R² = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R² = 0.60 and R² = 0.71, respectively).

Conclusions

Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.     

The Photochemistry Of 5-BrdCpdC,dCp5-BrdCpdA,and dCp5-BrdCdT in Aqueous Solution

We isolated an intrastrand crosslink product from the Pyrex-filtered UV light irradiation of 5-BrdCpdC in aqueous solution. ESI-MS, MS/MS, and 2-D nuclear Overhauser effect spectroscopy (NOESY) results showed that the C5 carbon atoms of the two cytosines are covalently bonded. The same cross-link product can also be induced from the similar UV irradiation of dCp5-BrdCpdA and dCp5-BrdCpdT.     

The effect of bisulfite on the dehalogenation of 5-chloro-, 5-bromo-, and 5-iodouracil

The 5-chloro-, bromo-, and iodo-analogs of uracil are dehalogenated in the presence of sodium bisulfite to yield 5,6 dihydrouracil-6-sulfonate as the final product. Under similar conditions, 5-fluorouracil adds bisulfite to yield 5-fluoro-5,6 dihydrouracil-6-sulfonate but is not dehalogenated. Ultraviolet absorption spectra of 5-bromouracil and 5-iodouracil reacting under pseudo first-order conditions with bisulfite indicate that dehalogenation proceeds via a pathway which has 5-halo-5,6-dihydrouracil-6-sulfonate and uracil as intermediates. In the case of 5-chlorouracil, the rate of bisulfite attack on the 6-position of the chlorouracil ring system is very slow relative to the rate of bisulfite addition to uracil. Hence, although dechlorination does occur, ultraviolet absorption spectra of reaction mixtures containing bisulfite and 5-chlorouracil do not reveal the uracil absorption peak observed with both 5-iodouracil and 5-bromouracil. Fluorine and proton nmr spectra indicate that bisulfite addition to 5-fluorouracil is stereoselective as is the case of bisulfite addition to uracil.     

The photochemistry of 5-BrdCpdC, dCp5-BrdCpdA, and dCp5-BrdCdT in aqueous solution

We isolated an intrastrand crosslink product from the Pyrex-filtered UV light irradiation of 5-BrdCpdC in aqueous solution. ESI-MS, MS/MS, and 2-D nuclear Overhauser effect spectroscopy (NOESY) results showed that the C5 carbon atoms of the two cytosines are covalently bonded. The same cross-link product can also be induced from the similar UV irradiation of dCp5-BrdCpdA and dCp5-BrdCpdT.