CLONING AND SEQUENCING OF THE ALLOPHYCOCYANIN GENES FROM SPIRULINA MAXIMA (CYANOPHYTA)

The genes coding for the α-and β-subunit of allophycocyanin (apcA and apcB) fromthe cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotidesequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The aminoacid sequence identities between S. maxima and S. platensis are 99 .4% for α subunit and 100% for βsubunit.

Subunit composition and chromophore content of R－phycocyanin and allophycocyanin from Porphyra haitanensis

R-phycocyanin (RPC) of Porphyra haitanensis (T. J. Chang et B. F. Zheng )' was chromatographed on Bio-Rex 70 column with urea solution (pH 3. 0) as an eluent, and a and β two subunits were isolated.Their molecular weights were determined on SDS-PAGE at 18 400 and 20 500, respetively,while those of a and β subunits of allophycocyanin (APC) at 18 800 and 19 700, respectively,and those of RPC and APC were at 117 000 and 122 000,respectively.Both the molar ratio of a and β subests of RPC and APC were 1:1, and the subunit composition was confirmed to be (αβ )3.It was ascertained that in RPC αsubunit contains one chromophore phycocyanobilin (PCB) and β subunit has one chromophore PCB and one phycoerythrobilin (PEB), while in APC both α and βsubunits contain one PCB.

Construction of energy transfer model of C-phycocyanin and allophycocyanin from Spirulina platensis

PHYCOBILIPROTEINS. which include phycoerythrin, phycocyanin, phycoerythroeyanin and allophycocyanin, are intensively coloured, highly fluorescent, water soluble chromoproteins. They are composed of at least two dissimilar polypeptides, α and β subunits, in a molar ratio of 1:1. The subunits possess linear tetrapyrrole prosthetic groups covalently linked to the primary structure via cysteine thioether linkages. In blue-green and red algae, phycobiliproteins occur in a supramolecular structure, the phycobilisome, located on the surface of thylakoid to

Excitation energy transfer mechanisms in allophycocyanin(Ⅰ)——Energy transfer mechanisms in monomer

The mechanisms of excitation energy transfer within allophycocyanin monomer with the theory of generalized master equation (GME) and the technique of time-resolved fluorescence anisotropic spectroscopy are studied. In the case of known information of its structure and spectra, the theory applied is based on the assumption that the coupling interaction between two chromophores is fairly weak. The theory correctly predicts the experimentlly observed rate for excitation energy transfer in allophycocyanin monomer. Based on the results, the energy transfer mechanism can be described as Frster and these processes cannot take place from the high vibrational levels of donor to acceptor.

Photocurrent generation by recombinant allophycocyanin trimer multilayer on TiO_2 electrode

Received 30 November 2012 Received in revised form 5 December 2012 Accepted 12 December 2012 Available online 21 January 2013

Preliminary crystallographic investigation of allophycocyanin from Porphyra yezoensis in cubic crystal form

THE phycobilisomes in blue-green algae and red algae are the functional units which absorb and transfer light energy. These supramolecular aggregates are attached on the outer surface of the thylakoid or photosynthetic membrane. Phycobilisomes absorb light in the range of 450—650 nm with an overall quantum efficiency of almost 100%, Electron microscopic studies showed that phycobilisomes consist of two distinct structure parts: the core and the rods.

Molecular replacement studies on crystal structure of allophycocyanin from red algae Porphyra yezoensis

Using the crystal structure of allophycocyanin from cyanobacterium Spirulina platensis (APC-SP) as a search model, the crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) has been studied by molecular replacement methods. The APC-PY crystals (Form 3) belong to the space group of R32, cell dimensions a = b = 10.53 nm, c = 18.94 nm, α =β = 90°, γ= 120°; there is one αβ monomer in each crystallographic asymmetric unit in the cell. The translation function search gave a unique peak with a correlation coefficient (Cc) of 67.0% and an R-factor of 36.1 % for reflection data from 1.0 to 0.4 nm. Using the results by molecular replacement, the initial model of APC-PY was built, and the coincidence of the chromophore in APC-PY initial model with its 2Fo-FC OMIT map further confirms the results by molecular replacement.

Computer simulation on the energy transfer processes in allophycocyanins

The allophycocyanin core is situated between the photosynthetic reaction center and the rods of a phycobilisome of algae and it accepts the excitation energy from the rods and then transfers the energy into the reaction center. Based on the crystal structure data and the spectra of the subunits in allophycocyanin, the energy transfer processes in the monomer and trimer of allophycocyanin were studied by the computer simulation technique. It was learnt from the simulation that the energy transfer kinetics between the two chromophores in an allophycocyanin monomer was very similar to that in a C\|phycocyanin monomer, while the energy transfer nature in an APC trimer was quite different from that in a C\|PC trimer. In a C\|phycocyanin trimer, the β 84 chromophores are the main fluorescent emitters, while the α 84 ones are the main fluorescent emitters in an APC trimer. This can be explained by their structure characteristics in phycobilisomes, that is, the basical unit of C\|phycocyanin in the rod of PBS is the hexamer while that of APC in the core of PBS is the trimer. The energy transfer from one trimer to another is most likely through the α 84 chromophores, from which it can be further deduced that one APC trimer approaches another in the face\|to\|face manner. The structural feature of APC is necessary to realize the energy transfer function in high efficiency.

Model of chromophore coupling in allophycocyanin aggregation

A new model is proposed to study the interaactions among chromphores in APC trimer. Using this model, the electronic transition frequencies are calculated. The results are in good agreement with the experimental.

钝顶节旋藻别藻蓝蛋白基因克隆、分析及重组表达蛋白荧光活性的研究

为探究蓝藻藻胆体核心色素蛋白-别藻蓝蛋白单个亚基的光学活性和功能,本研究克隆了钝顶节旋藻(Arthrospira platensis FACHB314)中的脱辅基别藻蓝蛋白基因apcAB及其亚基基因apcA和apcB。节旋藻基因组中apcAB全长共1056 bp,由apcA和apcB串联组成,中间间隔84 bp。其中,apcA和apcB基因的全长均为486 bp,且均编码161个氨基酸,预测分子量分别约为17.39和17.33 kDa,它们分别编码的α和β亚基均属于Globin_like超级家族,系统进化树分析显示其氨基酸序列在蓝藻和节旋藻中较为保守。将克隆出的亚基基因apcA、apcB分别同前期A.platensis FACHB314中获得的藻蓝胆素合成相关基因ho和pcyA以及色基裂合酶基因(cpcE、cpcF、cpcU、cpcS、cpcT)共同转化至大肠杆菌中,获得重组别藻蓝蛋白亚基菌株E.coli HPEFUSTA、E.coli HPEFUSTB。SDS-PAGE和Western Blot均显示重组菌株中已成功表达别藻蓝蛋白单亚基。荧光检测结果显示,在600 nm波长激发下,两株重组菌株在640 nm处均出现了别藻蓝蛋白特征荧光发射峰。这些结果表明在重组大肠杆菌中表达的脱辅基别藻蓝蛋白的单亚基可以和藻蓝胆素结合,形成有光学活性的别藻蓝蛋白,这为研究蓝藻中藻胆体的组装和功能提供有效的元件,为别藻蓝蛋白在医药、荧光检测方面的应用奠定了基础。

钝顶螺旋藻中藻蓝蛋白提取纯化新工艺

用０３ｍｍｏｌ／Ｌ的十二烷基苯磺酸钠提取钝顶螺旋藻中的藻蓝蛋白，提取率达到了９８％。再在硅藻土５４５柱上分级洗脱，进一步经ＤＥＡＥ—纤维素柱纯化，其纯度达到了４１（指可见区最大吸收与２８０ｎｍ处之比），最大吸收峰在６１９ｎｍ，室温荧光发射峰在６４３ｎｍ。

从一种富含藻胆蛋白的螺旋藻中大量提取和纯化藻蓝蛋白的研究

采用新鲜藻丝为原料和分段梯度盐析分离纯化钝顶螺旋藻 Sp(NS) - 90 0 2 0的藻蓝蛋白 (PC) ,经羟基磷灰石一次层析 ,能使提取的 PC的纯度大于普遍认可的标准。由于该工艺流程较为简单 ,适合藻蓝蛋白的大量生产。藻胆蛋白经 Sephadex凝胶过滤后的可达电泳纯度标准。经 SDS- PAGE测得 PC,APC的分子量分别约为 38,33k

基因重组别藻蓝蛋白对小鼠S_(180)肉瘤的抑制作用

研究基因重组别藻蓝蛋白 (RAPC)的抑瘤作用 ,给皮下接种S180 瘤细胞的小鼠 ,每天分别ig及ipRAPC 13 4mg/kg ,6 7mg/kg和 3 4mg/kg ,共 10d ,第 11d处死小鼠 ,称瘤重和胸腺重 ,计数白细胞。结果表明 ,RAPC对小鼠S180 肉瘤有显著抑制作用 ,瘤重抑制率在 45 %～ 64 %之间 ,ig及ip均有效 ,对荷瘤小鼠的胸腺指数和白细胞数量无明显影响。

坛紫菜别藻蓝蛋白α,β亚基基因的克隆和序列分析

以野生坛紫菜色素体DNA为模板，通过PCR扩增获得编码坛紫菜别藻蓝蛋白α亚基和β亚基的序列及两者之间的间隔序列，该序列全长为1055bp，其中编码α和β亚基的序列均为486bp，间隔序列为83bp，该序列与GenBank收录的其他4种红藻相关序列的同源性在75．6％～87．4％，与2种蓝藻的同源性分别为66．9％，68．5％，其中编码区同源性更高。

钝顶螺旋藻藻胆蛋白的分离、纯化及其理化特性

钝顶螺旋藻(Spirulina Platensis var.nanjingensis)一变异株的水溶性色素精提物,经固体硫酸铵沉淀,羟基磷灰石(HA)和Sephadex G-100柱层析后可分离、纯化出藻蓝蛋白(C-PC)和别藻蛋白(APC)。它们的纯度可分别达到AS 620/A_(277)=4.71;A_(650)/A_(270)=5.62。纯化后的C—PC和APC在聚丙烯酰胺凝胶电泳(PAGE)中仅见一条色带,其最大吸收峰分别在620nm和050nm。经12％的十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE),以及高效液相色谱(HPLC)分离,C—PC和APC均可分为α和β两个亚单位。两者的亚单位分子量分别为:C—PC—α,15000;C—PC—β,14500;APC—α,15000;APC—β,13500。依此推算,该藻的C—PC和APC的最小分子量应为29.5kD和28.5kD。经等电电泳法测定,其C—PC和APC的等电点分别在4.8和4.9。氨基酸组成和含量分析结果表明,除色氨酸(Try)未测外,c—PC含有14种氨基酸,APC含有15种氨基酸,两者都缺乏组氨酸(His)和脯氨酸(Pro),C—PC还缺少蛋氨酸(Met)。

盐生隐杆藻藻胆蛋白的分离纯化和特性研究

盐生隐杆藻的水溶性色素提取物经硫酸铵沉淀、DEAE SepharoseFastFlow、羟基磷灰石和SephacrylS 2 0 0柱层析 ,纯化出两种藻胆蛋白 :藻蓝蛋白 (C PC)和别藻蓝蛋白 (APC) ,纯度 (A62 0 /A2 80 )分别为 5 .5和 5 .0。紫外 可见分光光度计测得C PC和APC的最大可见吸收峰分别为 6 17nm和 6 5 1nm ,其室温荧光发射峰分别为 6 4 5nm和 6 6 4nm。SDS PAGE显示 ,C PC和APC均有α和β亚基。C PC的α和β亚基分子质量分别为 15 6 6 7u和 18197u ;APC的α和 β亚基分子质量分别为 14 894u和 17378u。根据SDS PAGE推算 ,C PC和APC单体的分子质量分别为 3386 4u和 32 2 72u ,质谱测得C PC和APC单体的分子质量分别为 380 14u和 36 4 32u。经等电聚焦测定C PC和APC的等电点分别约为 4 .6和 4 .8。氨基酸分析结果表明 ,C PC和APC分别含有 17种氨基酸。

江蓠属藻胆蛋白的研究──Ⅱ.龙须菜藻胆蛋白光谱特性和荧光动力学研究

本文以野生型和突变型龙须菜为材料，测定了完整藻体的室温吸收光谱、77K低温荧光发射光谱和叶绿素诱导荧光动力学曲线．野生型（Wt）和黄色突变体（Ye100）的室温吸收光谱有435、498、540、566、618、640和680nm7个吸收峰；绿色突变体Gr180和Gr184则只有除540nm以外的6个吸收峰．3种突变体的藻红蛋白含量均比野生型少，两种绿色突变体的藻蓝蛋白含量相对有所增加，别藻蓝蛋白含量变化不明显．77K低温荧光发射发谱（激发光分别为436、540和580nm）的结果表明：在发射峰的波长上，4种材料没有明显的区别，但Wt中PE吸收的光能传递效率比较低．叶绿素诱导荧光动力学曲线显示Wt的光系统Ⅱ活性、光系统Ⅱ原初光能转化效率及光合碳代谢活性都高于突变体．

几种重组别藻蓝蛋白的抗氧化活性

藻胆蛋白是某些藻类特有的捕光色素蛋白,其中天然别藻蓝蛋白、藻蓝蛋白的抗氧化活性已经被证明。实验以2,2-盐酸脒基丙烷(AAPH)为自由基生成者,应用藏红花素退色反应为检测清除氢过氧自由基的方法,探讨了在大肠杆菌(Escherichia coli)中表达并纯化的带有6×His(6×组氨酸)标签和带有麦芽糖结合蛋白(MBP)标签的重组别藻蓝蛋白及其α、β亚基的抗氧化活性。结果表明,带有His标签的6×Hisα-APC、6×Hisβ-APC和6×His-APC均显示出一定的清除氢过氧自由基的能力,其中6×Hisβ-APC清除氢过氧自由基的IC50值可达到27.2 mg/L,Ka/Kc(氢过氧自由基与重组别藻蓝蛋白和藏红花素反应的速度常数比)达到1.24,大于带有色素基团的天然APC清除氢过氧自由基能力(IC5057.5 mg/L);而带有MBP标签的重组别藻蓝蛋白亚基MBPα-APC和MBPβ-APC和MBP-APC(rAPC)则无明显的清除能力。结果首次证实了脱辅基蛋白具有清除氢过氧自由基能力,因而在天然藻胆蛋白中脱辅基蛋白是清除自由基的贡献者之一,这为进一步研究重组别藻蓝蛋白的抗肿瘤机理提供了线索。

海生红藻中藻蓝、别藻蓝蛋白的凝胶过滤分离

选用Sephadex G-150(G-150)、Sephadex G-200(G-200)、Sephacryl S-200(S-200)和Sephacryl S-300(S-300)四种凝胶对富含藻红蛋白的多管藻藻胆蛋白提取液进行过滤,并对所得藻蓝、别藻蓝蛋白组分做进一步纯化,优化建立了有效分离去除藻红蛋白、纯化制备藻蓝、别藻蓝蛋白组分的技术方法,即依次使用G-150、S-300和S-200凝胶过滤,所得样品中藻蓝和别藻蓝蛋白相对含量显著提高。并且根据实验现象,对凝胶柱的选择性、柱效、内水体积以及样品的蛋白组成等主要因素与凝胶过滤实际效果的相互关系进行了论述。

螺旋藻别藻蓝蛋白的纯化、理化特性与结晶

硫酸铵分级沉淀结合多种层析技术,从螺旋藻(SP-Dz)中纯化到电泳纯别藻蓝蛋白(Allophycocyanin,APC),纯度(A650/A280)达4·83。APC在30min内的荧光扫描曲线为直线;30min连续光照其相对荧光强度仍为原来的98%以上,其抗荧光淬灭能力强于同样条件下的藻蓝蛋白(PC)及荧光素TRITC。用悬滴气相扩散法培养获得了APC晶体。