Intergeneric conjugation in holomycin-producing marine Streptomyces sp strain M095

Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isotated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9 +/- 0.13 x 10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively. (c) 2006 Elsevier GmbH. All rights reserved.

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Heterologous expression and purification of recombinant allophycocyanin in marine Streptomyces sp isolate M097

Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10(-4) exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2-3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

六种重组别藻蓝蛋白的抗氧化活性研究

藻胆蛋白（ phycobiliprotein ）是某些藻类中一类重要的捕光色素蛋白，由脱辅基蛋白（ apoprotein ）和四吡咯结构的藻胆素（ phycocyanobilin ）共价结合组成，具有抗氧化和抗肿瘤等多种生物活性。本实验室利用两种载体pMAL-p2X和pET28a对APC及其亚基分别进行重组表达，产生了MBP-APC、MBP-αAPC（Mα）、MBP-βAPC（Mβ）、6×His-αAPC（Hα）、6×His-βAPC（Hβ）和6×His-APC（HAPC）六种重组别藻蓝蛋白。本研究将表达产物和天然APC一起进行抗氧化活性研究，以期筛选出分子量更小、活性更强的组分。 本研究通过以下模型比较了六种重组别藻蓝蛋白和天然别藻蓝蛋白在体外不同抗氧化模型中的活性氧清除作用： 1. 通过比较对体外纯化学体系产生的羟自由基的清除作用，发现MBP系列的重组别藻蓝蛋白组分对体外化学体系产生的羟自由基均有一定的清除能力，清除活性大小依次为：MαAPC、rAPC、MβAPC、HβAPC、HαAPC、HAPC、Native APC。 2. 通过比较对体外纯化学体系产生的氢过氧自由基的清除作用，发现带有MBP标签的重组别藻蓝蛋白组分除MβAPC 有很低的抗氧化活性外，MαAPC和rAPC均无明显的抗氧化活性；而带有His标签的HβAPC和HAPC均高于天然APC，且随着浓度的提高，清除能力随之提高，呈现良好的量效关系。 3. 通过比较对体外纯化学体系产生的超氧阴离子自由基的清除作用，发现除MβAPC和MαAPC外，不同浓度的天然及重组别藻蓝蛋白组分对超氧阴离子自由基均有一定程度的清除作用，且随着浓度的增加清除率随之增加。其中，rAPC和HβAPC对超氧阴离子自由基的清除效果最强，IC50值分别达到36.98和42.27μg/mL。 4. 通过比较对O2-损伤红细胞膜的保护作用发现，天然及6种重组别藻蓝蛋白及其亚基对O2-损伤红细胞膜的作用均无显著的抑制作用。 5. 通过比较对•OH诱导的脂质过氧化的保护作用发现，，在100~500μg/mL的剂量范围内， rAPC和HβAPC有显著的抑制脂质过氧化的作用，IC50值分别为230.50和217.35μg/mL；HAPC 和Native APC有弱抑制作用；而MαAPC、MβAPC和HαAPC则无明显抑制脂质过氧化的作用。 我们又对其中各组分在不同体系中的抗氧化效果进行比较后发现：不同的抗氧化体系中，各种重组别藻蓝蛋白组分的抗氧化活性虽然各不相同，但带有His标签的重组别藻蓝蛋白组分明显高于带有MBP标签的重组别藻蓝蛋白组分；其中，HβAPC对体外羟自由基、氢过氧自由基、超氧阴离子自由基以及生物互作产生的•OH均有较强的清除作用，有望开发成为新一代抗氧化剂。 本研究筛选出了具有高效抗氧化活性的新型重组别藻蓝蛋白HβAPC（海普克），分子量小，且抗氧化活性比雷普克显著，这为进一步探讨雷普克和海普克的抗肿瘤作用机理提供了资料。

紫球藻藻红蛋白和藻蓝蛋白共价交联物的构建及其与天然藻胆体特性的比较研究

用蔗糖密度梯度超速离心的方法从单细胞红藻---紫球藻（Porphyridium cruentum）中分离纯化了完整的藻胆体。光谱分析表明，藻胆体在可见光区域有四个吸收峰（545nm, 565nm, 618nm 和 650nm）和一个吸收肩（498nm）；荧光发射峰分别在680nm和595nm，二者的比值（λ_(680nm)~(em)/λ_(595nm)~(em)）可高达10以上，说明分离的藻胆体非常完整。非变性电泳结果显示，分离的藻胆体至少有三种颜色的条带，分别为：B－PE，R－PC和b－PE，APC可能含量很少而未见到。SDS－PAGE电泳鉴定至少有三条明显的带，为PE的α和β的重叠带，γ亚基和RPC的亚基。用羟基磷灰石层析和Sephadex G-200层析的方法分离到了高纯度的B－藻红蛋白、b-藻红蛋白和较纯的R－藻蓝蛋白。光谱检验发现，分离得到的藻胆蛋白均为天然态，其中藻红蛋白的荧光发射峰在580nm左右，藻蓝蛋白的荧光发射峰在640nm左右。SDS－PAGE电泳分析，B-藻红蛋白有两条带，分子量大约在20kD，31kD。小分子量的条带极宽，是因为α和β亚基的分子量相近，所以有重叠；大分子量的条带是γ亚基。相对应的是b-藻红蛋白只有20kD的条带，无γ亚基的条带。藻蓝蛋白有两条明显的条带，大约为16kD和20kD。用戊二醛共价交连的方法得了紫球藻B-藻红蛋白和R－藻蓝蛋白和共价结合体。吸收光谱表明，交联体和对照最明显的区别是紫外光区278nm的峰高增加并且蓝移到268nm；交联体的荧光发射光谱与对照相比，498nm激发产生的峰值在588nm（游离的PE的发射峰）和650nm（交联体中PC的发射峰），而对照只有590nm的发射峰，并且交联体在588nm的峰高明显低于对照。非变性电泳鉴定，发现有与对照不同的新的条带出现。这一切证明藻胆蛋白能量传递模型共价交联成功。人工构建的交联物和天然的藻胆体相比，对低离子强度和低温（4 ℃）更为稳定，但能量传递效率相对较低。

坛紫菜中藻胆蛋白和多糖的化学结构特征研究

本文对北移坛紫菜（Porphyra haitanensis）中的藻胆蛋白和多糖进行了深入的研究，并发现其化学结构特征与南方坛紫菜存在一定差异，其主要结果如下：坛紫菜的水溶性色素粗提物经过耱酸铵沉淀和羟基磷灰石（HA）柱层析后，分离到三种藻胆蛋白，即藻蓝蛋白（RPC）、藻红蛋白（RPE）和变藻蓝蛋白（APC）。在中性介质中，其吸收光谱和荧光发射光谱与文献报告基本一致，但在酸性（PH_3）或碱性（PH_(12)）介质中，吸收光谱有明显改变，原有的荧光性质也消失。RPC和APC只分离到一种聚集体，但RPE有两种不同的聚体，用Sephadex G-100凝胶过滤方法，测得藻胆蛋白的分子量分别为：RPC 117，000，APC 122，000，小分子RPE 34，000，大分子RPE 232，000。 而且RPE的两种聚集体的吸收光谱、荧光光谱以及等电点等物理性质都有所差异。对RPE的氨基酸分析结果表明，以天冬氨酸、丙氨酸和谷氨酸为主，且酸性氨基酸的含量大于碱性基酸的含量。用分级法对南北方两种坛紫菜的热水和冷水提取多糖进行了层析分级，用~(13)C-NMR和红外光谱研究了各级分的化学结构特征，南北方坛紫菜琼胶分别主要由用1.0M和0.5M NaCl从DEAE-Sephadex A50层析柱洗脱下的带电荷的琼胶分子组成，~(13)C-NMR谱图表明坛紫菜主要由琼胶糖结构及其生物前体（硫酸基结合在L-半乳糖的第6位碳上）构成，且甲基化琼胶糖在南方坛紫菜中的含量明显比北移坛紫菜高，这是坛紫菜由南方移植北方后的明显差异，这种差异对于进一步研究坛紫菜的生理学特点是有意义的。