Molecular cloning and characterization of a C‐type lectin in yellow catfish Tachysurus fulvidraco

This study represents the first report of a C‐type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino‐acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real‐time (qrt)‐PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration‐dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram‐positive and Gram‐negative bacteria in a calcium‐dependent manner. These results suggested that Ctl might be a C‐type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).     

Soluble interleukin‐1 receptor,a potential negative regulator of orange‐spotted grouper Epinephelus coioides interleukin‐1 system

In this study, the cDNA sequence encoding interleukin‐1 (Il‐1) receptor‐like protein of orange‐spotted grouper Epinephelus coioides was obtained. The newly identified sequence was named soluble type I Il‐1 receptor (sIl‐1rI) owing to its structural composition, which had two Ig‐like domains, lack of transmembrane region and the Toll/interleukin‐1 receptor (TIR) domain, similar to the brown rat Rattus norvegicus soluble Il‐1rI. In addition, sequence comparison and phylogenetic analysis indicated that E. coioides sequence had a closer relationship with Il‐1rI than Il‐1rII. Real‐time PCR revealed that sil‐1rI mRNA expression presented a process of decrease, restoration and increase in Cryptocaryon irritans‐infected E. coioides. The negative correlation between Il‐1β and sil‐1rI mRNA in C. irritans‐infected head‐kidney implied the potential negative regulatory role of sil‐1rI in E. coioides Il‐1 system. The leucocytes incubated with lipopolysaccharide or polyriboinosinic polyribocytidylic acid exhibited different expression profiles of sil‐1rI. Recombinant Il‐1β (rIl‐1β) protein was capable of inducing sil‐1rI mRNA under the concentration of 100 ng ml^?1, suggesting that high dosage or excess Il‐1β would stimulate the expression of sil‐1rI to maintain the homoeostasis of E. coioides Il‐1 system. For the first time, the role of teleost Il‐1rI in parasite infection has been identified, and soluble Il‐1r was found in fish.     

Purification and characterization of a D‐mannose specific lectin from the green marine alga,Bryopsis plumosa

A d ‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d ‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l ‐fucose (8 mM). d ‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.     

Loop‐mediated isothermal amplification assay targeting the blaCTX‐M9 gene for detection of extended spectrum β‐lactamase‐producing Escherichia coli and Klebsiella pneumoniae

Extended‐spectrum β‐lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β‐lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL‐producing bacteria is necessary for identification, prevention and treatment. Loop‐mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL‐producing bacteria by a LAMP technique. ESBLs‐producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double‐disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla_CTX‐M9 gene was designed based on bla_CTX‐M9 from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder‐like patterns of band sizes from 226 bp of the bla_CTX‐M9 DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla_CTX‐M9 was greater than that of the PCR method by at least 10,000‐fold. These results showed that the LAMP primers specifically amplified only the bla_CTX‐M9 gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.     

Recombinant expression and purification of the antimicrobial peptide magainin‐2

Magainin‐2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram‐positive and gram‐negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N‐terminal of a family III carbohydrate‐binding module fused to the linker sequence (LK‐CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2‐LK‐CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK‐CBM3. Its functionality was confirmed by testing its activity against gram‐negative bacteria. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

MOLECULAR CHARACTERIZATION OF THE LECTIN,BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)1

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5′ untranslated region (UTR) and 201 bp of 3′ UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C‐terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

Molecular cloning and analysis of a C‐type lectin from silkworm Bombyx mori

C‐type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non‐catalytic carbohydrate‐recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single‐CRD CTL, named C‐type lectin‐S2 (BmCTL‐S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL‐S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL‐S2 is expressed in a variety of immune‐related tissues, including hemocytes and fat body among others. BmCTL‐S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL‐S2) bound different bacterial cell wall components and bacterial cells. rBmCTL‐S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL‐S2 is a pattern‐recognition receptor with antibacterial activities.     

Isotopologue profiling of the listerial N‐metabolism

The nitrogen (N‐) sources and the relative contribution of a nitrogenous nutrient to the N‐pool of the gram‐positive pathogen Listeria monocytogenes are largely unknown. Therefore, ¹⁵N‐isotopologue profiling was established to study the N‐metabolism of L. monocytogenes. The pathogen was grown in a defined minimal medium supplemented with potential ¹⁵N‐labeled nutrients. The bacteria were harvested and hydrolysed under acidic conditions, and the resulting amino acids were analysed by GC‐MS, revealing ¹⁵N‐enrichments and isotopomeric compositions of amino acids. The differential ¹⁵N‐profiles showed the substantial and simultaneous usage of ammonium, glutamine, methionine, and, to a lower extent, the branched‐chain amino acids valine, leucine, and isoleucine for anabolic purposes, with a significant preference for ammonium. In contrast, arginine, histidine and cysteine were directly incorporated into proteins. L. monocytogenes is able to replace glutamine with ethanolamine or glucosamine as amino donors for feeding the core N‐metabolism. Perturbations of N‐fluxes caused by gene deletions demonstrate the involvement of ethanolamine ammonia lyase, and suggest a role of the regulator GlnK of L. monocytogenes distinct from that of Escherichia coli. The metabolism of nitrogenous nutrients reflects the high flexibility of this pathogenic bacterium in exploiting N‐sources that could also be relevant for its proliferation during infection.     

Progressive genome‐wide introgression in agricultural Campylobacter coli

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein‐coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST‐828 and ST‐1150 clonal complexes. The ST‐1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST‐828 complex bacteria have 10–11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross‐species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.     

Hemocyte-mediated phagocytosis and melanization in the mosquito<Emphasis Type="Italic"> Armigeres subalbatus</Emphasis> following immune challenge by bacteria

Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(–) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.This work was supported by NIH grant AI19769 to B.M.C. and NIH grant F31 AI50252 to J.F.H.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Dose‐dependent fate of GFP‐expressing Escherichia coli in the alimentary canal of adult house flies

The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe‐rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose‐dependent survival of bacteria in house flies. Flies were fed four different ‘doses’ of green fluorescent protein (GFP)‐expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post‐ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time‐points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time‐points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose‐fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune‐mediated. Understanding dose‐dependent bacterial survival in flies can help in predicting bacteria transmission potential.     

V‐antigen homologs in pathogenic gram‐negative bacteria

Gram‐negative bacteria cause many types of infections in animals from fish and shrimps to humans. Bacteria use Type III secretion systems (TTSSs) to translocate their toxins directly into eukaryotic cells. The V‐antigen is a multifunctional protein required for the TTSS in Yersinia and Pseudomonas aeruginosa. V‐antigen vaccines and anti‐V‐antigen antisera confer protection against Yersinia or P. aeruginosa infections in animal models. The V‐antigen forms a pentameric cap structure at the tip of the Type III secretory needle; this structure, which has evolved from the bacterial flagellar cap structure, is indispensable for toxin translocation. Various pathogenic gram‐negative bacteria such as Photorhabdus luminescens, Vibrio spp., and Aeromonas spp. encode homologs of the V‐antigen. Because the V‐antigens of pathogenic gram‐negative bacteria play a key role in toxin translocation, they are potential therapeutic targets for combatting bacterial virulence. In the USA and Europe, these vaccines and specific antibodies against V‐antigens are in clinical trials investigating the treatment of Yersinia or P. aeruginosa infections. Pathogenic gram‐negative bacteria are of great interest because of their ability to infect fish and shrimp farms, their potential for exploitation in biological terrorism attacks, and their ability to cause opportunistic infections in humans. Thus, elucidation of the roles of the V‐antigen in the TTSS and mechanisms by which these functions can be blocked is critical to facilitating the development of improved anti‐V‐antigen strategies.     

Syntheses,Receptor Bindings,in vitro and in vivo Stabilities and Biodistributions of DOTA‐Neurotensin(8–13) Derivatives Containing β‐Amino Acid Residues – A Lesson about the Importance of Animal Experiments

Neurotensin(8–13) (NTS(8–13)) analogs with C‐ and/or N‐terminal β‐amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1 – 6 ). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivatives, 6a , into a crystallographically identified receptor NTSR1 (Fig. 1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell‐membrane homogenates, while, with NTSR1‐exhibiting cancer tissues, affinities in the single‐digit nanomolar range can be observed (Table 2). Most of the β‐amino acid‐containing NTS(8–13) analogs (Table 1 and Fig. 2), including the ⁶⁸Ga complexes of the DOTA‐substituted ones ( 6 ; Figs. 2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two ⁶⁸Ga complexes (of 6a and 6b ) in HT29 tumor‐bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET‐imaging experiments with the tumor‐bearing mice (Fig. 6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10–15 min) of the two ⁶⁸Ga complexes shows that they are rapidly cleaved in the animals (Fig. 5).     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

PURIFICATION OF A SEX‐SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.