紫球藻藻红蛋白基因的克隆及表达
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摘要
藻红蛋白可作为天然色素应用于食品和化妆品工业,还可以作为光敏剂和荧光探针广泛应用于医药行业。本论文主要从紫球藻(Porphyridium cruentum)藻红蛋白亚基基因的克隆,该亚基基因在巴斯德毕赤酵母和大肠杆菌BL21中进行异源表达等方面开展研究。主要实验结果如下：
在本课题组吴义诚已克隆出藻红蛋白β亚基的基础上,利用LA-PCR技术扩增出紫球藻藻红蛋白β、α亚基及间隔区序列(GenBank登录号：HM535635),cDNA序列长1134 bp个碱基,包括p亚基编码区的534个碱基(编码177个氨基酸),105个碱基的间隔区以及a亚基编码区495个碱基(编码164个氨基酸)。cDNA序列排布顺序为5'UTR-peB-间隔区-peA,对其基因组结构分析表明,藻红蛋白(Phyeoerythrin, PE)亚基基因编码区无内含子序列。利用生物信息学的方法对α亚基的氨基酸序列,氨基酸组成,疏水作用,二级结构和三级结构等方面进行了初步的分析和预测。结果显示,预测的藻红蛋白α亚基结构与不同来源的藻红蛋白α亚基结构十分吻合,同时它的结构特征也满足于捕光蛋白的特性。
藻红蛋白α、p亚基分别亚克隆到毕赤酵母表达系统pPIC9K和大肠杆菌表达载体pET28a进行异源表达。重组的毕赤酵母表达载体pAO815-peA-peB、pPIC9K-peA分别转入Pichia pastoris GS 115,经甲醇诱导,SDS-PAGE电泳及DOT-BLOT分析,发现表达量过低。将藻红蛋白α、β亚基分别克隆到pET28a载体上,再分别转入E. coli BL21, IPTG诱导,WesternBlot分析有单一的条带出现,结果显示藻红蛋白α、β亚基在大肠杆菌表达系统中主要以包涵体的形式存在。
对peB的密码子偏好进行了分析,结果表明peB在密码子使用上存在显著的偏好性,偏爱于使用以A或T结尾的密码子。将藻红蛋白亚基基因与大肠杆菌、毕赤酵母基因组的密码子偏爱性进行比较,结果表明,藻红蛋白亚基基因密码子用法与大肠杆菌、毕赤酵母差异较大,若要实现该基因在以上宿主中的高效表达则需对部分密码子进行改造。
As a natural pigment, phycoerythrin can be used in food and cosmetics industries as well as widely applyed in the pharmaceutical industry for photosensitizer and fluorescent probes. In this paper, we cloned phycoerythrin (PE) subunit gene and then expressed in Pichia pastoris and Escherichia coli BL21 for heterologous expression. The results were as follows:
Group member Wu Yicheng has clonedβsubunits of phycoerythrin, base on his results, amplified phycoerythrinβ,αsubunit and intergenic region by LA-PCR, then sequenced(GenBank Accession Number:HM535635), cDNA sequence has 1134 bp bases, including the P subunit coding region of 534 bp (encoding 177 amino acids),105 bp spacer region and 495 bp a subunit (encoding 164 amino acids). cDNA sequences arranged in order of 5'UTR-peB-spacer region-peA. The analysis of genomic sequence showed that Porphyridium cruentum gene of PE has no introns. The primary structure, the secondary structure, and the three-dimensional structure of the PE a subunit in Porphyridium cruentum were predicted through the homologous peA with known structure in different data bases. The results suggested that the main structure of the a subunit of PE in Porphyridium cruentum was suited for the feature of light-harvesting protein.
The peA, peB cDNA were subcloned into pPIC9K vector and the recombinant plasmid was transformed into Pichia pastoris GS115. The peA, peB was overexpressed in Pichia pastoris GS115 after induction under the control of methanol, but the expression of peA, peB were low. Then, the encoding gene of peA, peB were inserted into the plasmid pPET28a and was successfully expressed in E. coli BL21 by IPTG.
Codon usage bias analysis shows that peA, peB have significant preference of the A or T ending codons. While compare to the E.coli and Pichia pastoris, the preference of peA, peB codon usage were significant different. If we want to increase the significantly efficiency of peA, peB gene expression, the partial codon in gene's open reading frame should be reconstituted.
在本课题组吴义诚已克隆出藻红蛋白β亚基的基础上,利用LA-PCR技术扩增出紫球藻藻红蛋白β、α亚基及间隔区序列(GenBank登录号：HM535635),cDNA序列长1134 bp个碱基,包括p亚基编码区的534个碱基(编码177个氨基酸),105个碱基的间隔区以及a亚基编码区495个碱基(编码164个氨基酸)。cDNA序列排布顺序为5'UTR-peB-间隔区-peA,对其基因组结构分析表明,藻红蛋白(Phyeoerythrin, PE)亚基基因编码区无内含子序列。利用生物信息学的方法对α亚基的氨基酸序列,氨基酸组成,疏水作用,二级结构和三级结构等方面进行了初步的分析和预测。结果显示,预测的藻红蛋白α亚基结构与不同来源的藻红蛋白α亚基结构十分吻合,同时它的结构特征也满足于捕光蛋白的特性。
藻红蛋白α、p亚基分别亚克隆到毕赤酵母表达系统pPIC9K和大肠杆菌表达载体pET28a进行异源表达。重组的毕赤酵母表达载体pAO815-peA-peB、pPIC9K-peA分别转入Pichia pastoris GS 115,经甲醇诱导,SDS-PAGE电泳及DOT-BLOT分析,发现表达量过低。将藻红蛋白α、β亚基分别克隆到pET28a载体上,再分别转入E. coli BL21, IPTG诱导,WesternBlot分析有单一的条带出现,结果显示藻红蛋白α、β亚基在大肠杆菌表达系统中主要以包涵体的形式存在。
对peB的密码子偏好进行了分析,结果表明peB在密码子使用上存在显著的偏好性,偏爱于使用以A或T结尾的密码子。将藻红蛋白亚基基因与大肠杆菌、毕赤酵母基因组的密码子偏爱性进行比较,结果表明,藻红蛋白亚基基因密码子用法与大肠杆菌、毕赤酵母差异较大,若要实现该基因在以上宿主中的高效表达则需对部分密码子进行改造。
As a natural pigment, phycoerythrin can be used in food and cosmetics industries as well as widely applyed in the pharmaceutical industry for photosensitizer and fluorescent probes. In this paper, we cloned phycoerythrin (PE) subunit gene and then expressed in Pichia pastoris and Escherichia coli BL21 for heterologous expression. The results were as follows:
Group member Wu Yicheng has clonedβsubunits of phycoerythrin, base on his results, amplified phycoerythrinβ,αsubunit and intergenic region by LA-PCR, then sequenced(GenBank Accession Number:HM535635), cDNA sequence has 1134 bp bases, including the P subunit coding region of 534 bp (encoding 177 amino acids),105 bp spacer region and 495 bp a subunit (encoding 164 amino acids). cDNA sequences arranged in order of 5'UTR-peB-spacer region-peA. The analysis of genomic sequence showed that Porphyridium cruentum gene of PE has no introns. The primary structure, the secondary structure, and the three-dimensional structure of the PE a subunit in Porphyridium cruentum were predicted through the homologous peA with known structure in different data bases. The results suggested that the main structure of the a subunit of PE in Porphyridium cruentum was suited for the feature of light-harvesting protein.
The peA, peB cDNA were subcloned into pPIC9K vector and the recombinant plasmid was transformed into Pichia pastoris GS115. The peA, peB was overexpressed in Pichia pastoris GS115 after induction under the control of methanol, but the expression of peA, peB were low. Then, the encoding gene of peA, peB were inserted into the plasmid pPET28a and was successfully expressed in E. coli BL21 by IPTG.
Codon usage bias analysis shows that peA, peB have significant preference of the A or T ending codons. While compare to the E.coli and Pichia pastoris, the preference of peA, peB codon usage were significant different. If we want to increase the significantly efficiency of peA, peB gene expression, the partial codon in gene's open reading frame should be reconstituted.
引文
[1]王长海.紫球藻及其应用研究[J].海洋通报.1998,17(003)：79-85.
[2]Brody M, Vatter AE. Observations on cellular structures of Porphyridium cruentum[J]. J Biophys Biochem Cytol.1959,5(2):289-294.
[3]Ramus J. The production of extracellular polysaccharide by the unicellular red alga Porphyridium aerugineum[J]. J. Phycol.1972,8:97-111.
[4]王明兹,施巧琴.紫球藻的培养与利用(综述)[J].亚热带植物科学.2001,30(002)：66-69.
[5]Gantt E, Conti S. The ultrastructure of Porphyridium cruentum[J]. The Journal of cell biology.1965,26(2):365.
[6]Schornstein K, Scott J. ULtrastructure of cell division in the unicellular red alga Porphyridium purpureum[J]. Canadian Journal of Botany.1982,60(1):85-97.
[7]Medcalf D, Scott J, Brannon J, et al. Some structural features and viscometric properties of the extracellular polysaccharide from Porphyridium cruentum[J]. Carbohydrate Research.1975,44(1):87-96.
[8]Heaney-Kieras J. Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum[J]. Carbohydrate Research.1976,52(1):169-177.
[9]Kost-Reyes E, Kost HP. The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments[J]. Eur J Biochem. 1979,102(1):83-91.
[10]Thepenier C, Chaumont D, Gudin C. Mass culture of Porphyridium cruentum:a multiproduct strategy for the biomass valorization[J]. Algal Biotechnology. 1988,413:C420.
[11]Rowan K. Photosynthetic pigments of algae[M]. Cambridge University Press, 1989:166-189.
[12]Bryant D, Glazer A, Eiserling F. Characterization and structural properties of the major biliproteins of Anabaena sp[J]. Archives of Microbiology.1976, 110(1):61-75.
[13]Glazer A. Phycobiliproteins-a family of valuable, widely used fluorophores[J]. Journal of Applied Phycology.1994,6(2):105-112.
[14]Schluchter W, Glazer A. Biosynthesis of phycobiliproteins in cyanobacteria[J]. The Phototrophic Prokaryotes.1999:83:C95.
[15]Beale S, Cornejo J. Biosynthesis of phycobilins. Ferredoxin-mediated reduction of biliverdin catalyzed by extracts of Cyanidium caldarium[J]. Journal of Biological Chemistry.1991,266(33):22328.
[16]Beale S, Cornejo J. Biosynthesis of phycobilins.15,16-Dihydrobiliverdin IX alpha is a partially reduced intermediate in the formation of phycobilins from biliverdin IX alpha[J]. Journal of Biological Chemistry.1991,266(33):22341.
[17]Beale S, Cornejo J. Biosynthesis of phycobilins.3 (Z)-phycoerythrobilin and 3 (Z)-phycocyanobilin are intermediates in the formation of 3 (E)-phycocyanobilin from biliverdin IX alpha[J]. Journal of Biological Chemistry.1991, 266(33):22333.
[18]Fairchild C, Zhao J, Zhou J, et al. Phycocyanin alpha-subunit phycocyanobilin lyase[J]. Proceedings of the National Academy of Sciences of the United States of America.1992,89(15):7017.
[19]Swanson R, Zhou J, Leary J, et al. Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment[J]. Journal of Biological Chemistry.1992,267(23):16146.
[20]Glazer A. Light guides. Directional energy transfer in a photosynthetic antenna[J]. Journal of Biological Chemistry.1989,264(1):1.
[21]MacColl R. Cyanobacterial Phycobilisomes*1[J]. Journal of structural biology. 1998,124(2-3):311-334.
[22]Glazer A. Phycobilisome:a macromolecular complex optimized for light energy transfer[J]. Biochimica et biophysica acta.1984,768(1):29-51.
[23]隋正红,张学成.藻红蛋白研究进展[J].海洋科学.1998,22(4)：24-27.
[24]周百成,郑舜琴,曾呈奎.几种绿藻,褐藻和红藻的吸收光谱的比较研究[J].植物学报.1974,16(2)：146-155.
[25]潘忠正,周百成,曾呈奎.青岛海产红藻R—藻红蛋白光谱特性的比较研究[J].海洋与湖沼.1987,18(5)：419-425.
[26]Klotz A, Glazer A. Characterization of the bilin attachment sites in R-phycoerythrin[J]. Journal of Biological Chemistry.1985,260(8):4856.
[27]Lagarias J, Klotz A, Dallas J, et al. Exclusive A-ring linkage for singly attached phycocyanobilins and phycoerythrobilins in phycobiliproteins. Absence of singly D-ring-linked bilins[J]. Journal of Biological Chemistry.1988,263(26):12977.
[28]梁栋材,常文瑞.R—藻红蛋白三维结构研究[J].生命科学.1998,10(005)：207-209.
[29]黄岩,秦利.红毛菜中两种不同分子量的R—藻红蛋白的性质比较[J].生物化学与生物物理学报：英文版.1993,25(004)：433-440.
[30]Scheer H, Kufer W. Conformational studies of phycocyanin from Spirulina platensis[J].1977.
[31]王广策,邓田,曾呈奎.藻胆蛋白的研究概况(Ⅱ)—藻胆蛋白的结构及其光谱特性[J].海洋科学.2000,24(003)：19-22.
[32]王盛.珊瑚藻藻红蛋白的分离纯化与相关特性.[福建农林大学博士学位论文].2002：9.
[33]Ficner R, Huber R. Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the y subunit[J]. European Journal of Biochemistry.1993,218(1):103-106.
[34]常文瑞,朱晋昌.0.5 nm分辨率多管藻R—藻红蛋白的晶体结构[J].自然科学进展：国家重点实验室通讯.1995,5(002)：181-190.
[35]张玉忠,周百成.海洋红藻R—藻胆蛋白的扫描隧道显微镜研究[J].电子显微学报.1997,16(006)：723-725.
[36]喻玲华,曾繁杰.海洋多管藻的R—藻红蛋白的亚基组成和发色团含量[J]. 生物化学与生物物理学报：英文版.1990,22(003)：221-227.
[37]程凌江,蒋丽金.条斑紫菜中R—藻红蛋白的纯化及其α和β亚基的分离与发色团含量[J].海洋与湖沼.1990,21(004)：337-342.
[38]武栋.藻胆蛋白裂合酶性质及催化功能研究.[华中科技大学博士学位论文].2005：4.
[39]李邵蓉,林惠民.Rhodosorus mairinus中藻红蛋白的纯化及其性质的研究[J].水生生物学报.1996,20(003)：257-264.
[40]Orta-Ramirez A, Merrill J, Smith D. pH affects the thermal inactivation parameters of R-phycoerythrin from Porphyra yezoensis[J]. Journal of food science.2000,65(6):1046-1050.
[41]Bennett A, Siegelman H. Bile pigments of plants[J]. The Porphyrins.1979, 6:493-520.
[42]张以芳,刘旭川.螺旋藻藻蓝蛋白提取及稳定性试验[J].云南大学学报：自然科学版.1999,21(003)：230-232.
[43]李建宏,邰子厚.极大螺旋藻藻蓝蛋白性质的研究[J].南京大学学报：自然科学版.1996,32(001)：59-63.
[44]伍华菊,蒋丽金.条斑紫菜中R—藻红蛋白的生化特性[J].生物化学与生物物理学报.1994,26(5)：491-498.
[45]Apt K, Metzner S, Grossman A. The y subunits of phycoerythrin from a red alga: position in phycobilisomes and sequence characterization[J]. Journal of Phycology.2001,37(1):64-70.
[46]马圣媛,王广策,孙海宝,et al.藻胆蛋白的研究概况Ⅲ.蓝藻藻胆蛋白合成的分子机制及调控[J].海洋科学.2002,26(008)：40-43.
[47]Thomas J, Passaquet C. Characterization of a phycoerythrin without y-Subunits from a unicellular Red Alga[J]. Journal of Biological Chemistry.1999, 274(4):2472.
[48]Sheng W, Fu-Di Z, Zu-Jian W, et al. Cloning and sequencing the y subunit of R-phycoerythrin from Corallina officinalis[J].植物学：英文版.2004,46(10)：1135-1140.
[49]Houmard J, Capuano V, Coursin T, et al. Genes encoding core components of the phycobilisome in the cyanobacterium Calothrix sp. strain PCC 7601:occurrence of a multigene family[J]. Journal of bacteriology.1988,170(12):5512.
[50]Zheng-hong S, Xue-cheng Z. Cloning and analysis of phycoerythrin genes inGracilaria Lemaneiformis (Rhodophyceae)[J]. Chinese Journal of Oceanology and Limnology.2000,18(1):42-46.
[51]Maid U, Valentin K, Zetsche K. The psbA-gene from a red alga resembles those from cyanobacteria and cyanelles[J]. Current genetics.1990,17(3):255-259.
[52]Apt K, Collier J, Grossman A. Evolution of the phycobiliproteins[J]. Journal of molecular biology.1995,248(1):79-96.
[53]钟伏弟.珊瑚藻R-藻红蛋白的分子生物学.[福建农林大学硕士学位论文].2003：12.
[54]Apt KE, Hoffman NE, Grossman AR. The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae[J]. J Biol Chem.1993, 268(22):16208-16215.
[55]Arciero D, Bryant D, Glazer A. In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin[J]. Journal of Biological Chemistry. 1988,263(34):18343.
[56]Fairchild C, Glazer A. Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin[J]. Journal of Biological Chemistry.1994,269(46):28988.
[57]Scheer H, Zhao K. Biliprotein maturation:the chromophore attachment[J]. Molecular microbiology.2008,68(2):263-276.
[58]Zhou J, Gasparich G, Stirewalt V, et al. The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants[J]. Journal of Biological Chemistry.1992,267(23):16138.
[59]Fairchild C, Glazer A. Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase[J]. Journal of Biological Chemistry.1994,269(12):8686.
[60]Wilbanks S, Glazer A. Rod structure of a phycoerythrin II-containing phycobilisome. I. Organization and sequence of the gene cluster encoding the major phycobiliprotein rod components in the genome of marine Synechococcus sp. WH8020[J]. Journal of Biological Chemistry.1993,268(2):1226.
[61]Kahn K, Mazel D, Houmard J, et al. A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis[J]. J Bacteriol.1997,179(4):998-1006.
[62]Kufer W, H gner A, Eberlein M, et al. Structure and molecular evolution of the gene cluster encoding proteins of the rod substructure of the phycobilisome from the cyanobacterium Mastigocladus laminosus[J]. Gene Bank.1991,75599.
[63]Shen G, Saunee N, Gallo E, et al. Identification of novel phycobiliprotein lyases in cyanobacteria. Niederman R, Blankenship R, Frank H,et al. in PS 2004 light-harvesting systems workshop, Quebec, Canada,2004, International society of photosynthesis research:14-15.
[64]Cobley J, Clark A, Weerasurya S, et al. CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR)[J]. Molecular microbiology.2002,44(6):1517-1531.
[65]Shen G, Schluchter W, Bryant D. Biogenesis of phycobiliproteins. I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocaynobilin lyase specific for γ-phycocyanin and allophycocyanin subunits[J]. Journal of Biological Chemistry.2008.
[66]Saunee N, Williams S, Bryant D, et al. Biogenesis of Phycobiliproteins:Ⅱ. CpcS-Ⅰ and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 of beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002. [J]. The Journal of Biological Chemistry.2008, 283(12):7513.
[67]Zhao KH, Su P, Tu JM, et al. Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins[J]. Proc Natl Acad Sci U S A.2007,104(36):14300-14305.
[68]Zhao K, Su P, Li J, et al. Chromophore attachment to phycobiliprotein beta-subunits:phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp. PCC7120[J]. Journal of Biological Chemistry.2006,281:8573-8581
[69]Shen G, Saunee NA, Williams SR, et al. Identification and characterization of a new class of bilin lyase:the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002[J]. J Biol Chem.2006,281(26):17768-17778.
[70]Nakamura Y, Kaneko T, Sato S, et al. Complete genome structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids[J]. DNA research. 2003,10(4):137.
[71]Zhao K, Zhang J, Tu J, et al. Lyase Activities of CpcS-and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin β-subunits[J]. Journal of Biological Chemistry.2007,282(47):34093.
[72]Wiethaus J, Busch AW, Kock K, et al. CpeS is a lyase specific for attachment of 3Z-PEB to Cys82 of {beta}-phycoerythrin from Prochlorococcus marinus MED4[J]. J Biol Chem.2010,285(48):37561-37569.
[73]Apt KE, Grossman AR. Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta)[J]. Plant molecular biology.1993,21(1):27-38.
[74]Roell MK, Morse DE. Organization, expression and nucleotide sequence of the operon encoding R-phycoerythrin α and β subunits from the red alga Polysiphonia boldii[J]. Plant molecular biology.1993,21(1):47-58.
[75]闫国良,杨听林,徐双悦,et al.坛紫菜藻红蛋白α,β亚基基因的克隆和序列分析[J].海洋学报.2007,29(002)：167-172.
[76]隋正红,张学诚.龙须菜藻红蛋白亚基基因的克隆及其在大肠杆菌中的表达[J].高技术通讯.2001,11(007)：14-19.
[77]Bryant D, Dubbs J, Fields P, et al. Expression of phycobiliprotein genes in Escherichia coli[J]. FEMS Microbiology Letters.1985,29(3):343-349.
[78]林凡,秦松.乳糖诱导重组别藻蓝蛋白基因在大肠杆菌中的表达[J].海洋科学.2005,29(011)：22-27.
[79]杨听林,左正宏,蔡嘉力,et al.坛紫菜别藻蓝蛋白β亚基的表达及鉴定[J].厦门大学学报：自然科学版.2007,46(002)：244-247.
[80]温若冰.重组藻红蛋白的表达及活性研究和链状亚历山大藻钙调蛋白基因的克隆及表达分析.[中国海洋大学硕士学位论文].2007：9-26
[81]Tooley AJ, Glazer AN. Biosynthesis of the Cyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-{alpha} subunit in a heterologous host[J]. Journal of bacteriology.2002,184(17):4666.
[82]Ge B, Sun H, Feng Y, et al. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli[J]. Journal of bioscience and bioengineering.2009, 107(3):246-249.
[83]Isailovic D, Sultana I, Phillips GJ, et al. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits[J]. Analytical biochemistry.2006,358(1):38-50.
[84]隋正红.龙须菜藻红蛋白基因的研究.[中国海洋大学博士学位论文].2001：28
[85]Vergara J, Niell F. effects of nitrate availability and irradiance on internal nitrogen constituents in corallina elongata (rhodophyta) [J]. Journal of Phycology.1993, 29(3):285-293.
[86]Eriksen N, Iversen J. Photosynthetic pigments as nitrogen stores in the cryptophyte alga Rhodomonas sp[J]. Journal of marine biotechnology.1995, 3:193-195.
[87]Rodriguez H, Rivas J, Guerrero M, et al. Enhancement of phycobiliprotein production in nitrogen-fixing cyanobacteria[J]. Journal of Biotechnology.1991, 20(3):263-270.
[88]Kaixian Q, Franklin M, Borowitzka M. The study for isolation and purification of R-phycoerythrin from a red alga[J]. Applied Biochemistry and Biotechnology. 1993,43(2):133-139.
[89]Cowles T, Desiderio R, Neuer S. In situ characterization of phytoplankton from vertical profiles of fluorescence emission spectra[J]. Marine Biology.1993, 115(2):217-222.
[90]Maeda H, Kawai A, Tilzer M. The water bloom of cyanobacterial picoplankton in Lake Biwa, Japan[J]. Hydrobiologia.1992,248(2):93-103.
[91]Subramaniam A, Carpenter E. An empirically derived protocol for the detection of blooms of the marine cyanobacterium Trichodesmium using CZCS imagery[J]. International Journal of Remote Sensing.1994,15(8):1559-1569.
[92]Caruso G, Zaccone R. Distribution of Synechococcus sp. and Synechococcus bacillaris in the waters of the straits of Magellan (April 1995-early austral autumn)[J]. The New microbiologica.1998,21(4):379-389.
[93]Almasri N, Iturraspe J, Braylan R. CD 10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles[J]. Archives of pathology & laboratory medicine.1998,122(6):539-544.
[94]Bhalgat M, Haugland R, Pollack J, et al. Green-and red-fluorescent nanospheres for the detection of cell surface receptors by flow cytometry[J]. Journal of immunological methods.1998,219(1-2):57-68.
[95]李冠武,王广策.R—藻红蛋白介导的光敏反应诱导小鼠S180细胞凋亡的研究[J].汕头大学医学院学报.2002,15(003)：133-135.
[96]仵小南,周百成.藻胆蛋白和高等植物类囊体之间的能量传递[J].植物学报：英文版.1993,35(009)：657-663.
[97]仵小南,周百成.藻胆蛋白与绿藻光合膜之间的激发能传递[J].海洋学报.1993,15(003)：94-100.
[98]Apt KE, Collier JL, Grossman AR. Evolution of the phycobiliproteins[J]. Journal of molecular biology.1995,248(1):79-96.
[99]Betz M. One century of protein crystallography:the phycobiliproteins[J]. Biological chemistry.378(3-4):167.
[100]Ritter S, Hiller R, Wrench P, et al. Purification, crystallization and preliminary X-ray analysis of a phycourobilin-containing phycoerythrin[J]. Protein and Peptide Letters.1997,4:69-74.
[101]吴义诚.紫球藻藻红蛋白p亚基基因的克隆及其在毕赤酵母中的表达.[福建师范大学硕士学位论文].2009：31-44.
[102]Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production[J]. Journal of molecular recognition.2005,18(2):119-138.
[103]Sharp PM, Tuohy TMF, Mosurski KR. Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes[J], Nucleic acids research. 1986,14(13):5125.
[104]Mansur M, Cabello C, Hernandez L, et al. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris[J]. Biotechnology letters. 2005,27(5):339-345.
[105]Beauvais A, Monod M, Debeaupuis JP, et al. Biochemical and antigenie characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus[J]. Journal of Biological Chemistry.1997,272(10):6238.
[106]Isailovic D, Sultana I, Phillips GJ, et al. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits[J]. Anal Biochem.2006,358(1):38-50.
[2]Brody M, Vatter AE. Observations on cellular structures of Porphyridium cruentum[J]. J Biophys Biochem Cytol.1959,5(2):289-294.
[3]Ramus J. The production of extracellular polysaccharide by the unicellular red alga Porphyridium aerugineum[J]. J. Phycol.1972,8:97-111.
[4]王明兹,施巧琴.紫球藻的培养与利用(综述)[J].亚热带植物科学.2001,30(002)：66-69.
[5]Gantt E, Conti S. The ultrastructure of Porphyridium cruentum[J]. The Journal of cell biology.1965,26(2):365.
[6]Schornstein K, Scott J. ULtrastructure of cell division in the unicellular red alga Porphyridium purpureum[J]. Canadian Journal of Botany.1982,60(1):85-97.
[7]Medcalf D, Scott J, Brannon J, et al. Some structural features and viscometric properties of the extracellular polysaccharide from Porphyridium cruentum[J]. Carbohydrate Research.1975,44(1):87-96.
[8]Heaney-Kieras J. Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum[J]. Carbohydrate Research.1976,52(1):169-177.
[9]Kost-Reyes E, Kost HP. The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments[J]. Eur J Biochem. 1979,102(1):83-91.
[10]Thepenier C, Chaumont D, Gudin C. Mass culture of Porphyridium cruentum:a multiproduct strategy for the biomass valorization[J]. Algal Biotechnology. 1988,413:C420.
[11]Rowan K. Photosynthetic pigments of algae[M]. Cambridge University Press, 1989:166-189.
[12]Bryant D, Glazer A, Eiserling F. Characterization and structural properties of the major biliproteins of Anabaena sp[J]. Archives of Microbiology.1976, 110(1):61-75.
[13]Glazer A. Phycobiliproteins-a family of valuable, widely used fluorophores[J]. Journal of Applied Phycology.1994,6(2):105-112.
[14]Schluchter W, Glazer A. Biosynthesis of phycobiliproteins in cyanobacteria[J]. The Phototrophic Prokaryotes.1999:83:C95.
[15]Beale S, Cornejo J. Biosynthesis of phycobilins. Ferredoxin-mediated reduction of biliverdin catalyzed by extracts of Cyanidium caldarium[J]. Journal of Biological Chemistry.1991,266(33):22328.
[16]Beale S, Cornejo J. Biosynthesis of phycobilins.15,16-Dihydrobiliverdin IX alpha is a partially reduced intermediate in the formation of phycobilins from biliverdin IX alpha[J]. Journal of Biological Chemistry.1991,266(33):22341.
[17]Beale S, Cornejo J. Biosynthesis of phycobilins.3 (Z)-phycoerythrobilin and 3 (Z)-phycocyanobilin are intermediates in the formation of 3 (E)-phycocyanobilin from biliverdin IX alpha[J]. Journal of Biological Chemistry.1991, 266(33):22333.
[18]Fairchild C, Zhao J, Zhou J, et al. Phycocyanin alpha-subunit phycocyanobilin lyase[J]. Proceedings of the National Academy of Sciences of the United States of America.1992,89(15):7017.
[19]Swanson R, Zhou J, Leary J, et al. Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment[J]. Journal of Biological Chemistry.1992,267(23):16146.
[20]Glazer A. Light guides. Directional energy transfer in a photosynthetic antenna[J]. Journal of Biological Chemistry.1989,264(1):1.
[21]MacColl R. Cyanobacterial Phycobilisomes*1[J]. Journal of structural biology. 1998,124(2-3):311-334.
[22]Glazer A. Phycobilisome:a macromolecular complex optimized for light energy transfer[J]. Biochimica et biophysica acta.1984,768(1):29-51.
[23]隋正红,张学成.藻红蛋白研究进展[J].海洋科学.1998,22(4)：24-27.
[24]周百成,郑舜琴,曾呈奎.几种绿藻,褐藻和红藻的吸收光谱的比较研究[J].植物学报.1974,16(2)：146-155.
[25]潘忠正,周百成,曾呈奎.青岛海产红藻R—藻红蛋白光谱特性的比较研究[J].海洋与湖沼.1987,18(5)：419-425.
[26]Klotz A, Glazer A. Characterization of the bilin attachment sites in R-phycoerythrin[J]. Journal of Biological Chemistry.1985,260(8):4856.
[27]Lagarias J, Klotz A, Dallas J, et al. Exclusive A-ring linkage for singly attached phycocyanobilins and phycoerythrobilins in phycobiliproteins. Absence of singly D-ring-linked bilins[J]. Journal of Biological Chemistry.1988,263(26):12977.
[28]梁栋材,常文瑞.R—藻红蛋白三维结构研究[J].生命科学.1998,10(005)：207-209.
[29]黄岩,秦利.红毛菜中两种不同分子量的R—藻红蛋白的性质比较[J].生物化学与生物物理学报：英文版.1993,25(004)：433-440.
[30]Scheer H, Kufer W. Conformational studies of phycocyanin from Spirulina platensis[J].1977.
[31]王广策,邓田,曾呈奎.藻胆蛋白的研究概况(Ⅱ)—藻胆蛋白的结构及其光谱特性[J].海洋科学.2000,24(003)：19-22.
[32]王盛.珊瑚藻藻红蛋白的分离纯化与相关特性.[福建农林大学博士学位论文].2002：9.
[33]Ficner R, Huber R. Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the y subunit[J]. European Journal of Biochemistry.1993,218(1):103-106.
[34]常文瑞,朱晋昌.0.5 nm分辨率多管藻R—藻红蛋白的晶体结构[J].自然科学进展：国家重点实验室通讯.1995,5(002)：181-190.
[35]张玉忠,周百成.海洋红藻R—藻胆蛋白的扫描隧道显微镜研究[J].电子显微学报.1997,16(006)：723-725.
[36]喻玲华,曾繁杰.海洋多管藻的R—藻红蛋白的亚基组成和发色团含量[J]. 生物化学与生物物理学报：英文版.1990,22(003)：221-227.
[37]程凌江,蒋丽金.条斑紫菜中R—藻红蛋白的纯化及其α和β亚基的分离与发色团含量[J].海洋与湖沼.1990,21(004)：337-342.
[38]武栋.藻胆蛋白裂合酶性质及催化功能研究.[华中科技大学博士学位论文].2005：4.
[39]李邵蓉,林惠民.Rhodosorus mairinus中藻红蛋白的纯化及其性质的研究[J].水生生物学报.1996,20(003)：257-264.
[40]Orta-Ramirez A, Merrill J, Smith D. pH affects the thermal inactivation parameters of R-phycoerythrin from Porphyra yezoensis[J]. Journal of food science.2000,65(6):1046-1050.
[41]Bennett A, Siegelman H. Bile pigments of plants[J]. The Porphyrins.1979, 6:493-520.
[42]张以芳,刘旭川.螺旋藻藻蓝蛋白提取及稳定性试验[J].云南大学学报：自然科学版.1999,21(003)：230-232.
[43]李建宏,邰子厚.极大螺旋藻藻蓝蛋白性质的研究[J].南京大学学报：自然科学版.1996,32(001)：59-63.
[44]伍华菊,蒋丽金.条斑紫菜中R—藻红蛋白的生化特性[J].生物化学与生物物理学报.1994,26(5)：491-498.
[45]Apt K, Metzner S, Grossman A. The y subunits of phycoerythrin from a red alga: position in phycobilisomes and sequence characterization[J]. Journal of Phycology.2001,37(1):64-70.
[46]马圣媛,王广策,孙海宝,et al.藻胆蛋白的研究概况Ⅲ.蓝藻藻胆蛋白合成的分子机制及调控[J].海洋科学.2002,26(008)：40-43.
[47]Thomas J, Passaquet C. Characterization of a phycoerythrin without y-Subunits from a unicellular Red Alga[J]. Journal of Biological Chemistry.1999, 274(4):2472.
[48]Sheng W, Fu-Di Z, Zu-Jian W, et al. Cloning and sequencing the y subunit of R-phycoerythrin from Corallina officinalis[J].植物学：英文版.2004,46(10)：1135-1140.
[49]Houmard J, Capuano V, Coursin T, et al. Genes encoding core components of the phycobilisome in the cyanobacterium Calothrix sp. strain PCC 7601:occurrence of a multigene family[J]. Journal of bacteriology.1988,170(12):5512.
[50]Zheng-hong S, Xue-cheng Z. Cloning and analysis of phycoerythrin genes inGracilaria Lemaneiformis (Rhodophyceae)[J]. Chinese Journal of Oceanology and Limnology.2000,18(1):42-46.
[51]Maid U, Valentin K, Zetsche K. The psbA-gene from a red alga resembles those from cyanobacteria and cyanelles[J]. Current genetics.1990,17(3):255-259.
[52]Apt K, Collier J, Grossman A. Evolution of the phycobiliproteins[J]. Journal of molecular biology.1995,248(1):79-96.
[53]钟伏弟.珊瑚藻R-藻红蛋白的分子生物学.[福建农林大学硕士学位论文].2003：12.
[54]Apt KE, Hoffman NE, Grossman AR. The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae[J]. J Biol Chem.1993, 268(22):16208-16215.
[55]Arciero D, Bryant D, Glazer A. In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin[J]. Journal of Biological Chemistry. 1988,263(34):18343.
[56]Fairchild C, Glazer A. Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin[J]. Journal of Biological Chemistry.1994,269(46):28988.
[57]Scheer H, Zhao K. Biliprotein maturation:the chromophore attachment[J]. Molecular microbiology.2008,68(2):263-276.
[58]Zhou J, Gasparich G, Stirewalt V, et al. The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants[J]. Journal of Biological Chemistry.1992,267(23):16138.
[59]Fairchild C, Glazer A. Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase[J]. Journal of Biological Chemistry.1994,269(12):8686.
[60]Wilbanks S, Glazer A. Rod structure of a phycoerythrin II-containing phycobilisome. I. Organization and sequence of the gene cluster encoding the major phycobiliprotein rod components in the genome of marine Synechococcus sp. WH8020[J]. Journal of Biological Chemistry.1993,268(2):1226.
[61]Kahn K, Mazel D, Houmard J, et al. A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis[J]. J Bacteriol.1997,179(4):998-1006.
[62]Kufer W, H gner A, Eberlein M, et al. Structure and molecular evolution of the gene cluster encoding proteins of the rod substructure of the phycobilisome from the cyanobacterium Mastigocladus laminosus[J]. Gene Bank.1991,75599.
[63]Shen G, Saunee N, Gallo E, et al. Identification of novel phycobiliprotein lyases in cyanobacteria. Niederman R, Blankenship R, Frank H,et al. in PS 2004 light-harvesting systems workshop, Quebec, Canada,2004, International society of photosynthesis research:14-15.
[64]Cobley J, Clark A, Weerasurya S, et al. CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR)[J]. Molecular microbiology.2002,44(6):1517-1531.
[65]Shen G, Schluchter W, Bryant D. Biogenesis of phycobiliproteins. I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocaynobilin lyase specific for γ-phycocyanin and allophycocyanin subunits[J]. Journal of Biological Chemistry.2008.
[66]Saunee N, Williams S, Bryant D, et al. Biogenesis of Phycobiliproteins:Ⅱ. CpcS-Ⅰ and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 of beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002. [J]. The Journal of Biological Chemistry.2008, 283(12):7513.
[67]Zhao KH, Su P, Tu JM, et al. Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins[J]. Proc Natl Acad Sci U S A.2007,104(36):14300-14305.
[68]Zhao K, Su P, Li J, et al. Chromophore attachment to phycobiliprotein beta-subunits:phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp. PCC7120[J]. Journal of Biological Chemistry.2006,281:8573-8581
[69]Shen G, Saunee NA, Williams SR, et al. Identification and characterization of a new class of bilin lyase:the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002[J]. J Biol Chem.2006,281(26):17768-17778.
[70]Nakamura Y, Kaneko T, Sato S, et al. Complete genome structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids[J]. DNA research. 2003,10(4):137.
[71]Zhao K, Zhang J, Tu J, et al. Lyase Activities of CpcS-and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin β-subunits[J]. Journal of Biological Chemistry.2007,282(47):34093.
[72]Wiethaus J, Busch AW, Kock K, et al. CpeS is a lyase specific for attachment of 3Z-PEB to Cys82 of {beta}-phycoerythrin from Prochlorococcus marinus MED4[J]. J Biol Chem.2010,285(48):37561-37569.
[73]Apt KE, Grossman AR. Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta)[J]. Plant molecular biology.1993,21(1):27-38.
[74]Roell MK, Morse DE. Organization, expression and nucleotide sequence of the operon encoding R-phycoerythrin α and β subunits from the red alga Polysiphonia boldii[J]. Plant molecular biology.1993,21(1):47-58.
[75]闫国良,杨听林,徐双悦,et al.坛紫菜藻红蛋白α,β亚基基因的克隆和序列分析[J].海洋学报.2007,29(002)：167-172.
[76]隋正红,张学诚.龙须菜藻红蛋白亚基基因的克隆及其在大肠杆菌中的表达[J].高技术通讯.2001,11(007)：14-19.
[77]Bryant D, Dubbs J, Fields P, et al. Expression of phycobiliprotein genes in Escherichia coli[J]. FEMS Microbiology Letters.1985,29(3):343-349.
[78]林凡,秦松.乳糖诱导重组别藻蓝蛋白基因在大肠杆菌中的表达[J].海洋科学.2005,29(011)：22-27.
[79]杨听林,左正宏,蔡嘉力,et al.坛紫菜别藻蓝蛋白β亚基的表达及鉴定[J].厦门大学学报：自然科学版.2007,46(002)：244-247.
[80]温若冰.重组藻红蛋白的表达及活性研究和链状亚历山大藻钙调蛋白基因的克隆及表达分析.[中国海洋大学硕士学位论文].2007：9-26
[81]Tooley AJ, Glazer AN. Biosynthesis of the Cyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-{alpha} subunit in a heterologous host[J]. Journal of bacteriology.2002,184(17):4666.
[82]Ge B, Sun H, Feng Y, et al. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli[J]. Journal of bioscience and bioengineering.2009, 107(3):246-249.
[83]Isailovic D, Sultana I, Phillips GJ, et al. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits[J]. Analytical biochemistry.2006,358(1):38-50.
[84]隋正红.龙须菜藻红蛋白基因的研究.[中国海洋大学博士学位论文].2001：28
[85]Vergara J, Niell F. effects of nitrate availability and irradiance on internal nitrogen constituents in corallina elongata (rhodophyta) [J]. Journal of Phycology.1993, 29(3):285-293.
[86]Eriksen N, Iversen J. Photosynthetic pigments as nitrogen stores in the cryptophyte alga Rhodomonas sp[J]. Journal of marine biotechnology.1995, 3:193-195.
[87]Rodriguez H, Rivas J, Guerrero M, et al. Enhancement of phycobiliprotein production in nitrogen-fixing cyanobacteria[J]. Journal of Biotechnology.1991, 20(3):263-270.
[88]Kaixian Q, Franklin M, Borowitzka M. The study for isolation and purification of R-phycoerythrin from a red alga[J]. Applied Biochemistry and Biotechnology. 1993,43(2):133-139.
[89]Cowles T, Desiderio R, Neuer S. In situ characterization of phytoplankton from vertical profiles of fluorescence emission spectra[J]. Marine Biology.1993, 115(2):217-222.
[90]Maeda H, Kawai A, Tilzer M. The water bloom of cyanobacterial picoplankton in Lake Biwa, Japan[J]. Hydrobiologia.1992,248(2):93-103.
[91]Subramaniam A, Carpenter E. An empirically derived protocol for the detection of blooms of the marine cyanobacterium Trichodesmium using CZCS imagery[J]. International Journal of Remote Sensing.1994,15(8):1559-1569.
[92]Caruso G, Zaccone R. Distribution of Synechococcus sp. and Synechococcus bacillaris in the waters of the straits of Magellan (April 1995-early austral autumn)[J]. The New microbiologica.1998,21(4):379-389.
[93]Almasri N, Iturraspe J, Braylan R. CD 10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles[J]. Archives of pathology & laboratory medicine.1998,122(6):539-544.
[94]Bhalgat M, Haugland R, Pollack J, et al. Green-and red-fluorescent nanospheres for the detection of cell surface receptors by flow cytometry[J]. Journal of immunological methods.1998,219(1-2):57-68.
[95]李冠武,王广策.R—藻红蛋白介导的光敏反应诱导小鼠S180细胞凋亡的研究[J].汕头大学医学院学报.2002,15(003)：133-135.
[96]仵小南,周百成.藻胆蛋白和高等植物类囊体之间的能量传递[J].植物学报：英文版.1993,35(009)：657-663.
[97]仵小南,周百成.藻胆蛋白与绿藻光合膜之间的激发能传递[J].海洋学报.1993,15(003)：94-100.
[98]Apt KE, Collier JL, Grossman AR. Evolution of the phycobiliproteins[J]. Journal of molecular biology.1995,248(1):79-96.
[99]Betz M. One century of protein crystallography:the phycobiliproteins[J]. Biological chemistry.378(3-4):167.
[100]Ritter S, Hiller R, Wrench P, et al. Purification, crystallization and preliminary X-ray analysis of a phycourobilin-containing phycoerythrin[J]. Protein and Peptide Letters.1997,4:69-74.
[101]吴义诚.紫球藻藻红蛋白p亚基基因的克隆及其在毕赤酵母中的表达.[福建师范大学硕士学位论文].2009：31-44.
[102]Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production[J]. Journal of molecular recognition.2005,18(2):119-138.
[103]Sharp PM, Tuohy TMF, Mosurski KR. Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes[J], Nucleic acids research. 1986,14(13):5125.
[104]Mansur M, Cabello C, Hernandez L, et al. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris[J]. Biotechnology letters. 2005,27(5):339-345.
[105]Beauvais A, Monod M, Debeaupuis JP, et al. Biochemical and antigenie characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus[J]. Journal of Biological Chemistry.1997,272(10):6238.
[106]Isailovic D, Sultana I, Phillips GJ, et al. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits[J]. Anal Biochem.2006,358(1):38-50.