藻红蓝蛋白β亚基体内重组及其色谱分析

藻胆蛋白是蓝藻中的捕光蛋白,其生物合成的重要一步是藻胆色素与脱辅基蛋白的连接.大多数藻胆色素的正确连接都需要结合位点专一和对色素的构象有选择性的裂合酶来催化完成,但是这方面的报道不是很多.藻红蓝蛋白由两个亚基组成,β亚基(简称β-PEC)含171个氨基酸残基及两个辅基色素藻蓝胆素(简称PCB),分别在Cys-84和Cys-155位以硫醚键共价相连.通过同源性分析获得的由编号为alr0617基因编码的蛋白为藻红蓝蛋白β亚基(β-PEC)中的Cys-84与PCB的连接的催化酶.为了研究层理鞭枝藻藻红蓝蛋白(PEC)β亚基(β-PEC)中藻蓝胆素(PCB)与脱辅基蛋白的连接机制,通过体内重组方式得到色素蛋白PCB-PecB(C155I),分析表明该色素蛋白与β-PEC的吸收光谱和荧光光谱一致.酸性尿素变性实验证明得到的色素蛋白中的藻蓝胆素PCB没有被破坏.使用胃蛋白酶对天然藻红蓝色素蛋白和重组藻红蓝色素蛋白进行相同条件的水解并得到各自的色素肽,高效液相色谱分析表明这两种色素肽相同,由此证明了编号为alr0617基因编码的蛋白质能催化PCB与PecB(C155I)正确共价偶联.     

高碘酸钠和戊二醛交联法构建的R-藻红蛋白-C-藻蓝蛋白交联物能量传递效率的比较研究

从单细胞蓝藻钝顶螺旋藻中纯化C-藻蓝蛋白,从海洋红藻多管藻纯化R-藻红蛋白.分别用高碘酸钠氧化法和戊二醛法将二者共价连接为R-藻红蛋白-C-藻蓝蛋白交联物,再用Sephadex G-200柱层析纯化.光谱分析表明,用两种方法构建的共价交联物都可以将激发能从R-藻红蛋白传递到C-藻蓝蛋白.二者相比,高碘酸钠氧化法构建的共价交联物的能量传递效率更高.     

藻红蓝蛋白α-亚基脱辅基蛋白与藻蓝胆素的重组

利用在大肠杆菌中表达的藻红蓝蛋白α－亚基脱辅基蛋白与藻蓝胆素PCB重组,吸收光谱、荧光光谱和高效可逆光化学性质分析表明,藻红蓝蛋白α－亚基脱辅基蛋白与藻蓝胆素直接重组,生成的胆素蛋白中辅基色素仍为藻蓝胆素;而藻红蓝蛋白α-亚基脱辅基蛋白与藻蓝胆素在藻红蓝蛋白α－亚基重组酶（pecE和pecF基因的表达产物）催化下重组,生成的胆素蛋白中辅基色素转变为藻紫胆素,并具有高效可逆光化学特性。     

抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。     

R─藻红蛋白三维结构研究

藻类的捕光系统是由棒状的藻胆体构成的,藻胆体又是由藻红蛋白、藻蓝蛋白和变藻蓝蛋白组成的。光能从藻红蛋白传递到藻蓝蛋白,再传递到变藻蓝蛋白,最后传递到光反应中心,其传递效率接近100％。R─藻红蛋白是藻红蛋白的一种;它是多亚基,超大分子量的蛋白色素复合物。为了阐明光能传递的机理,我们使用X─射线晶体学的方法,对取自多管藻的R─藻红蛋白的三维结构进行了长期的研究并取得了一系列进展。首先,我们使用多对同晶置换法获得了R─藻红蛋白5A分辨率的结构,随后经过努力又获得了R─藻红蛋白2．8A中分辨率和1．9A高分辨率的结构,并首次解析了藻尿胆素的三维结构。我们通过对R─藻红蛋白三维结构的详细分析,对光能传递中的一些重要问题提出了自己的看法。该结构为我国有关藻类捕光蛋白的系列研究和光合作用机制研究打下了非常重要的基础。     

鱼腥藻PCC7120藻蓝蛋白α亚基体内重组研究

为了研究藻蓝蛋白α亚基的生物合成途径,通过构建相容的3种重组质粒pETDuet-cpcA、pCOLADuet-cpcE-cpcF和pACYCDuet-ho1-pcyA,将裂合酶基因cpcE和cpcF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻蓝蛋白α亚基基因cpcA共同转入大肠杆菌BL21(DE3)。通过色素蛋白锌电泳和光谱检测表明产生了生物活性的CpcA-PCB。成功实现了大肠杆菌内藻蓝蛋白α亚基84位半胱氨酸残基与PCB的连接。而在裂合酶基因cpcE和cpcF不转入大肠杆菌的情况下,大肠杆菌内只有0.2%的CpcA-PCB产生。以上研究为进一步在大肠杆菌内合成天然的藻蓝蛋白奠定了基础。     

B-藻红蛋白和R-藻红蛋白γ亚基的分离、特性及其在分子中的空间位置分析

紫球藻 (Porphyridiumcruentum)B 藻红蛋白和多管藻 (Polysiphoniaurceo lata)R -藻红蛋白经蛋白酶K部分酶切消化后 ,分离得到近似天然态的γ亚基 ,并且对它的光谱特性以及在藻红蛋白分子中的空间位置进行了研究 .酶解动力学分析表明γ亚基位于R- 藻红蛋白和B -藻红蛋白六聚体 (αβ) ₆的中央空洞中 .分离的γ亚基上藻红胆素的吸收峰位于 5 89nm ,荧光发射峰位于 6 2 0nm ,与藻蓝蛋白的吸收峰重叠 ,有助于藻胆体中藻红蛋白与藻蓝蛋白分子间高效能量传递 .     

条斑紫菜藻红、藻蓝蛋白α和β亚基基因序列测定及分析

为了获得江苏吕泗的条斑紫菜藻红蛋白α(Pea)和β(Peb)亚基、藻 蓝蛋白α(Pca)和β(Pcb)亚基基因的DNA序列,本文对其基因进行了克隆、 序列测定及分析.根据已发表的基因序列(DQ666487.1)设计引物,对提取自 条斑紫菜叶状体的DNA进行PCR和电泳鉴定,产物经测序证实获得藻红蛋白 序列1 357 bp (HM008263.1)和藻蓝蛋白序列1 335 bp (HM008262.1).上述 两段序列与已报道的条斑紫菜相关序列(DQ666487.1)同源性均为99％,与 其它几种紫菜相关序列同源性为88％～98％.两段序列均采用多顺反子转录 策略,排布顺序为5′Untranslated Regions(UTR)- Peb -间隔区- Pea -3 ′UTR 和 5′UTR- Pcb -间隔区- Pca -3′UTR.文中对基因翻译所得氨基 酸序列做了理化参数、功能位点及空间构型的预测.基于对序列开放阅读框 、启动子、Shine-Dalgarno (SD)序列的分析,本文对条斑紫菜分类地位进 行了讨论.     

鱼腥藻PCC7120藻蓝蛋白α亚基体内重组研究

为了研究藻蓝蛋白α亚基的生物合成途径,通过构建相容的3种重组质粒pETDuet-cpcA、pCOLADuet-cpcE-cpcF和pACYCDuet-ho1-pcyA,将裂合酶基因cpcE和cpcF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻蓝蛋白α亚基基因cpcA共同转入大肠杆菌BL21(DE3)。通过色素蛋白锌电泳和光谱检测表明产生了生物活性的CpcA-PCB。成功实现了大肠杆菌内藻蓝蛋白α亚基84位半胱氨酸残基与PCB的连接。而在裂合酶基因cpcE和cpcF不转入大肠杆菌的情况下,大肠杆菌内只有0.2%的CpcA-PCB产生。以上研究为进一步在大肠杆菌内合成天然的藻蓝蛋白奠定了基础。     

Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His(6)-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys(82) of CpeA. The CpeS bilin lyase ligated PEB at both Cys(82) and Cys(139) of CpeA but very inefficiently; the yield of PEB ligated at Cys(82) was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys(80) of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.     

CpeY is a phycoerythrobilin lyase for cysteine 82 of the phycoerythrin I α-subunit in marine Synechococcus

Marine Synechococcus are widespread in part because they are efficient at harvesting available light using their complex antenna, or phycobilisome, composed of multiple phycobiliproteins and bilin chromophores. Over 40% of Synechococcus strains are predicted to perform a type of chromatic acclimation that alters the ratio of two chromophores, green-light–absorbing phycoerythrobilin and blue-light–absorbing phycourobilin, to optimize light capture by phycoerythrin in the phycobilisome. Lyases are enzymes which catalyze the addition of bilin chromophores to specific cysteine residues on phycobiliproteins and are involved in chromatic acclimation. CpeY, a candidate lyase in the model strain Synechococcus sp. RS9916, added phycoerythrobilin to cysteine 82 of only the α subunit of phycoerythrin I (CpeA) in the presence or absence of the chaperone-like protein CpeZ in a recombinant protein expression system. These studies demonstrated that recombinant CpeY attaches phycoerythrobilin to as much as 72% of CpeA, making it one of the most efficient phycoerythrin lyases characterized to date. Phycobilisomes from a cpeY⁻ mutant showed a near native bilin composition in all light conditions except for a slight replacement of phycoerythrobilin by phycourobilin at CpeA cysteine 82. This demonstrates that CpeY is not involved in any chromatic acclimation-driven chromophore changes and suggests that the chromophore attached at cysteine 82 of CpeA in the cpeY⁻ mutant is ligated by an alternative phycoerythrobilin lyase. Although loss of CpeY does not greatly inhibit native phycobilisome assembly in vivo, the highly active recombinant CpeY can be used to generate large amounts of fluorescent CpeA for biotechnological uses.     

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.     

Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility.

Assembly of holophytochrome in the plant cell requires covalent attachment of the linear tetrapyrrole chromophore precursor, phytochromobilin, to a unique cysteine in the nascent apoprotein. In this investigation we compare chromophore analogs with the natural chromophore precursor for their ability to attach covalently to recombinant oat apophytochrome and to form photoactive holoproteins. Ethylidene-containing analogs readily form covalent adducts with apophytochrome, whereas chromophores lacking this double bond are poor substrates for attachment. Kinetic measurements establish that although the chromophore binding site on apophytochrome is best tailored to phytochromobilin, apophytochrome will accommodate the two analogs with modified D-rings, phycocyanobilin and phycoerythrobilin. The phycocyanobilin-apophytochrome adduct is photoactive and undergoes a light-induced protein conformational change similar to the native holoprotein. By contrast, the phycoerythrobilin adduct is locked into a photochemically inactive protein conformation that is similar to the red light-absorbing Pr form of phytochrome. These results support the hypothesis that the photoconversion from Pr to Pfr, the far red light- absorbing form of phytochrome, involves the photoisomerization of the C15 double bond. Knowledge gained from these studies provides impetus for rational design of chromophore analogs whose insertion into apophytochrome should elicit profound changes in light-mediated plant growth and development.     

A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis.

Pigment mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon is characterized by constitutive synthesis of the phycobiliprotein phycoerythrin due to insertional inactivation of the rcaC regulatory gene by endogenous transposon Tn5469. Whereas the parental strain Fd33 harbors five genomic copies of Tn5469, cells of strain FdR1 harbor six genomic copies of the element; the sixth copy in FdR1 is localized to the rcaC gene. Electroporation of FdR1 cells yielded secondary pigment mutant strains FdR1E1 and FdR1E4, which identically exhibited the FdR1 phenotype with significantly reduced levels of phycoerythrin. In both FdR1E1 and FdR1E4, a seventh genomic copy of Tn5469 was localized to the cpeY gene of the sequenced but phenotypically uncharacterized cpeYZ gene set. This gene set is located downstream of the cpeBA operon which encodes the alpha and beta subunits of phycoerythrin. Complementation experiments correlated cpeYZ activity to the phenotype of strains FdR1E1 and FdR1E4. The predicted CpeY and CpeZ proteins share significant sequence identity with the products of homologous cpeY and cpeZ genes reported for Pseudanabaena sp. strain PCC 7409 and Synechococcus sp. strain WH 8020, both of which synthesize phycoerythrin. The CpeY and CpeZ proteins belong to a family of structurally related cyanobacterial proteins that includes the subunits of the CpcE/CpcF phycocyanin alpha-subunit lyase of Synechococcus sp. strain PCC 7002 and the subunits of the PecE/PecF phycoerythrocyanin alpha-subunit lyase of Anabaena sp. strain PCC 7120. Phycobilisomes isolated from mutant strains FdR1E1 and FdR1E4 contained equal amounts of chromophorylated alpha and beta subunits of phycoerythrin at 46% of the levels of the parental strain FdR1. These results suggest that the cpeYZ gene products function in phycoerythrin synthesis, possibly as a lyase involved in the attachment of phycoerythrobilin to the alpha or beta subunit.     

Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity

Cryptophytes are among the few eukaryotes employing phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to cyanobacterial PBP that are organized in membrane-associated phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin 545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. The assembly of PBPs in cryptophytes involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of the eukaryotic S-type PBP lyase GtCPES. We show GtCPES-mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP β-subunit (PmCpeB) of Prochlorococcus marinus MED4. On the basis of the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB), most likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colored complex in vitro and produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on these findings, we postulate that this single amino acid residue has a crucial role for bilin binding specificity of S-type phycoerythrin lyases but additional factors regulate handover to the target protein.     

Biogenesis of phycobiliproteins: I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phyococyanobilin lyase specific for beta-phycocyanin and allophycocyanin subunits

Phycobilin lyases covalently attach phycobilin chromophores to apo-phycobiliproteins (PBPs). Genome analyses of the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 identified three genes, denoted cpcS-I, cpcU, and cpcV, that were possible candidates to encode phycocyanobilin (PCB) lyases. Single and double mutant strains for cpcS-I and cpcU exhibited slower growth rates, reduced PBP levels, and impaired assembly of phycobilisomes, but a cpcV mutant had no discernable phenotype. A cpcS-I cpcU cpcT triple mutant was nearly devoid of PBP. SDS-PAGE and mass spectrometry demonstrated that the cpcS-I and cpcU mutants produced an altered form of the phycocyanin (PC) beta subunit, which had a mass approximately 588 Da smaller than the wild-type protein. Some free PCB (mass = 588 Da) was tentatively detected in the phycobilisome fraction purified from the mutants. The modified PC from the cpcS-I, cpcU, and cpcS-I cpcU mutant strains was purified, and biochemical analyses showed that Cys-153 of CpcB carried a PCB chromophore but Cys-82 did not. These results show that both CpcS-I and CpcU are required for covalent attachment of PCB to Cys-82 of the PC beta subunit in this cyanobacterium. Suggesting that CpcS-I and CpcU are also required for attachment of PCB to allophycocyanin subunits in vivo, allophycocyanin levels were significantly reduced in all but the CpcV-less strain. These conclusions have been validated by in vitro experiments described in the accompanying report (Saunée, N. A., Williams, S. R., Bryant, D. A., and Schluchter, W. M. (2008) J. Biol. Chem. 283, 7513-7522). We conclude that the maturation of PBP in vivo depends on three PCB lyases: CpcE-CpcF, CpcS-I-CpcU, and CpcT.     

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.     

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.