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Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes)
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e4, 2015 www.elsevier.com/locate/jbiosc
Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes) Yoshimitsu Takakura,1, *, x Kozue Sofuku,1 Masako Tsunashima,1 and Shigeru Kuwata2 Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan1 and Graduate School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan2 Received 21 April 2015; accepted 5 September 2015 Available online xxx
A biotin-binding protein with a low isoelectric point (pI), which minimizes electrostatic non-specific binding to substances other than biotin, is potentially valuable. To obtain such a protein, we screened hundreds of mushrooms, and detected strong biotin-binding activity in the fruit bodies of Lentinula edodes, shiitake mushroom. Two cDNAs, each encoding a protein of 152 amino acids, termed lentiavidin 1 and lentiavidin 2 were cloned from L. edodes. The proteins shared sequence identities of 27%e49% with other biotin-binding proteins, and many residues that directly associate with biotin in streptavidin were conserved in lentiavidins. The pI values of lentiavidin 1 and lentiavidin 2 were 3.9 and 4.4, respectively; the former is the lowest pI of the known biotin-binding proteins. Lentiavidin 1 was expressed as a tetrameric protein with a molecular mass of 60 kDa in an insect cell-free expression system and showed biotin-binding activity. Lentiavidin 1, with its pI of 3.9, has a potential for broad applications as a novel biotin-binding protein. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biotin-binding protein; Cell-free extract; Isoelectric point; Lentinula edodes; Non-specific binding]
Biotin-binding proteins have been found in a diverse array of organisms. For example, in vertebrates, avidin (1) and avidinrelated proteins (AVR1, AVR2, AVR3, AVR4/5, AVR6, AVR7) (2) were found in the chicken (Gallus gallus), xenavidin (3) in Xenopus tropicalis, and zebavidin (4) in Danio rerio. In bacteria, streptavidin (5) was found in Streptomyces avidinii, bradavidin (6) and bradavidin II (7) in Bradyrhizobium japonicum, rhizavidin (8) in Rhizobium etli, and burkavidin (9) in Burkholderia pseudomallei. In fungi, tamavidin 1 and tamavidin 2 (10) were found in tamogitake mushroom (Pleurotus cornucopiae). Avidin and streptavidin have been recognized for a long time and are well characterized. The dissociation constant (Kd) of biotin is 6 1016 M for avidin and 4 1014 M for streptavidin (11). The extraordinarily high affinities of the avidin- and streptavidin-biotin interactions are of great scientific value and have been exploited in diverse applications in medicine, biochemistry, and biotechnology (12,13). Among biotin-binding proteins, tamavidin 2 is unusual in that can be expressed at high levels in its soluble form in Escherichia coli, and it has a high affinity for biotin (10). Tamavidin 2 could, therefore, be produced industrially in E. coli and engineered to exhibit distinct characteristics such as a lower isoelectric point (14), reversible biotin-binding (15), or extremely high thermal stability (16). The detection or separation of biotinylated biomolecules from crude cell extracts is one of the most important applications of biotin-binding proteins, where the non-specific binding of proteins
* Corresponding author. Tel.: þ81 285 34 2655; fax: þ81 285 25 4460. E-mail address: [email protected] (Y. Takakura). x Present address: Leaf Tobacco Research Center, Japan Tobacco, Inc., 1900 Idei, Oyama, Tochigi 323-080, Japan.
to substances other than biotin can be extremely detrimental. In this regard, both avidin and streptavidin may show undesirable characteristics. Avidin, which is a basic glycoprotein, has shown a high level of non-specific binding to various biological components at physiological pH, resulting in high background levels (17). This high non-specific binding is thought to be due to its high isoelectric point (pI > 10). Although streptavidin has a lower pI (6.1e7.5), it contains an Arg-Tyr-Asp (RYD) tripeptide that apparently mimics the Arg-Gly-Asp (RGD) cell adhesion domain of fibronectin (18), thereby causing background binding in histochemical and cytochemical applications. The charge of a biotin-binding protein largely participates in non-specific binding to biological macromolecules. Since many biological macromolecules including DNA, RNA, acidic proteins or biomembranes are minus-charged in physiological pH, a biotinbinding protein with a lower pI value, which is minus-charged in such pH, may be effective to reduce the electrostatic chargedependent non-specific binding, and therefore be of value in various applications. Indeed, the mutant avidin Avm-pI4.7, with an acidic pI of 4.7, showed low non-specific binding to DNA and cells (17). A mutant tamavidin 2 with a pI of 5.8e6.2 also showed lower non-specific binding to DNA and glycoproteins from human sera compared with wild-type tamavidin 2 with a pI of 7.4e7.5 (14). Since mutations introduce the risk of disturbing the biotin-binding activity, an alternative approach could be to modify the proteins chemically or enzymatically, but such modification introduces the risk of creating heterogenous molecular species. Therefore, searching for novel biotin-binding proteins from unstudied sources has become an attractive approach. We screened various kinds of mushrooms for such proteins and here report the identification of
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
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two avidin-like proteins with pIs of 3.9 and 4.4, respectively in shiitake mushroom (Lentinula edodes).
J. BIOSCI. BIOENG.,
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MATERIALS AND METHODS Extraction of proteins and nucleic acids Mushrooms including L. edodes and P. cornucopiae were purchased from local grocery stores. Total soluble proteins were extracted in 0.1 M HEPES-KOH pH 7.5, precipitated by using ammonium sulfate (75% saturate), then dissolved in and dialyzed against 20 mM HEPES pH 7.5 according to the procedure described by Takakura and Kuwata (19). Genomic DNA was isolated by the standard SDS-phenol method, and mRNA was purified according to the procedure described by Takakura and Kuwata (19). Biotin-binding assay The procedure for non-denaturing SDS-PAGE and staining of biotin-binding activity with biotinylated horseradish peroxidase (HRP) was described previously by Takakura et al. (16). Briefly, the protein sample was dissolved in 1 SDS gel-loading buffer (20) without reducing agent at room temperature for 10 min, separated by SDS-PAGE on a 10% gel, and then blotted onto a PVDF membrane (Millipore). The membrane was blocked with Trisbuffered saline (TBS) containing 3% BSA and 0.2% Tween 20, incubated in TBS containing 3% BSA with biotinylated HRP (Vector Laboratories) (1:1000 dilution) at room temperature for 1 h, and washed three times with TBS containing 0.2% Tween 20. The biotin-binding proteins were visualized by using ECL Detection Reagents (Amersham Biosciences). Streptavidin from S. avidinii (60 kDa as a tetramer, from Sigma) was used as a control. cDNA cloning PCR was performed with L. edodes genomic DNA as a template. The 50-ml reaction contained 100 ng of genomic DNA and 5 pmol of each degenerate primer in 1 GC buffer (TakaraBio). The forward primer was 50 -GGN ACI TGG TAT/C AAT/C G/CAA/G C/TTN GG-30 and the reverse primer was 50 -TAT/C TGN CCI G/CA/TC CAN GTN GT-30 , where N represents A, T, G, or C, and I represents inosine. Ex-taq (0.5 U) (TakaraBio) was incubated with Taq Start Antibody (Clontech) at room temperature for 10 min and then added to the reaction, which then underwent one cycle of 96 C for 3 min, 40 cycles of 96 C for 1 min, 50 C for 1 min and 72 C for 1 min, and one cycle of 72 C for 6 min. Amplified DNA was cloned into the pCR2.1Topo vector (Invitrogen) and sequenced. The PCR product was used as a hybridization probe to screen the L edodes cDNA library, which was constructed from 5 mg of mRNA by using a lZAP cDNA synthesis kit (Stratagene). cDNA clones were rescued as pBluescript plasmids from l phages by using in vivo excision according to the manufacturer’s instructions. DNA and amino acid sequences were analyzed by using GENETYX-WIN ver.8 software (Genetyx Co.). Cell-free protein synthesis The PCR product was amplified from each cDNA clone (leav 1 or leav2) by using the primers 50 -ATGGCTCCTACGACATTGACATCGAGGCAA-30 and 50 -TGATCAGGATCCCTACGCAATCTCAGCACGAG-30 for leav 1 or the primers 50 -ATGGCTCCTACGACATTGACATCAAGGCAA-30 and 50 -CAATGAGGATCCTTACGCAGTCTCAGCACGAA-30 for leav2. The termini of the products were filled in by using the Klenow fragment of DNA polymerase I (TakaraBio). Each product was then digested with BamHI and cloned into the expression vector pTD1 (Shimadzu) predigested with EcoRV and BamHI. RNA was synthesized from 10 mg of the linearized expression vector by using the T7 RiboMAX Express Large Scale RNA Production System (Promega) in a 200-ml reaction. Protein was synthesized from 1 mg of the RNA by using Transdirect insect cell (DTTþ) (Shimadzu) in a 1-ml reaction, which consisted of 500-ml of insect cell extract, 300-ml of reaction buffer, 20-ml of 4 mM methionine, 200-ml of RNA at 25 C for 5 h, according to the manufacturer’s instructions. A protein synthesis reaction without RNA was run as a negative control. A 50-ml scale synthesis was performed by using the FluoroTect GreenLys in vitro Translation Labeling System (Promega), and protein expression was verified by means of SDS-PAGE and fluorescence detection. Biotin-binding assay with fluorescent biotin Binding to biotin-4fluroescein was assayed according to the protocol described by Kada et al. (21) and Takakura et al. (10). Briefly, various quantities of the cell-free extracts were incubated with biotin-4-fluorescein (Molecular Probe, 25 pmol per 200-ml assay) at room temperature for 30 min. Fluorescence was measured by using an Infinite M200 microplate reader (Tecan).
RESULTS AND DISCUSSION Biotin-binding activity in L. edodes Hundreds of mushroom species were screened for biotin-binding activities, which were assayed after total soluble proteins were separated by nondenaturing SDS-PAGE and immobilized onto a membrane. This assay method is based on the characteristic of biotin-binding proteins that they retain their activities (16) as well as their oligomeric structures (22) in non-denaturing SDS-PAGE in which the heat-denature of protein sample is omitted. This characteristic seems to come from the property of biotin-binding
60 kDa
60 kDa
FIG. 1. Biotin-binding activity from mushrooms. Proteins from Lentinula edodes (Lane 2) and Pleurotus cornucopiae (Lane 1), and streptavidin (Lane 3) were separated on nondenaturing SDS-PAGE and transferred onto a PVDF membrane. Biotin-binding activity was then visualized by using a biotinylated peroxidase.
proteins that their inter-subunit associations are tolerant to SDS, but not to heat-denature. A clear signal of approximately 60 kDa was detected in the extract from L. edodes (Fig. 1), indicating that a biotin-binding protein was present in L. edodes. The size of the biotin-binding protein from L. edodes was similar to those of tamavidins from P. cornucopiae (10) and streptavidin. Some other mushrooms were also positive in the screen, but the signals were not as strong as that in L. edodes. We therefore focused on the protein from L. edodes in this study. Cloning of cDNAs for biotin-binding proteins from L. edodes Degenerate primers based on consensus sequences among the amino acid sequences of three biotin-binding proteinsdstreptavidin, tamavidin 1, and tamavidin 2dwere designed (Fig. 2). A DNA segment was amplified from the genomic DNA of L. edodes by PCR using these primers. The sequence was similar to those of known biotin-binding proteins. By using the PCR product as a probe, we obtained eight cDNA clones from the screen of 88,000 plaques in the L. edodes cDNA library. The eight cDNA clones were sequenced and classified into two types. The longest clones of both types encoded polypeptides consisting of 152 residues (Fig. 2). We termed the encoded proteins lentiavidins (L. edodes avidin). Lentiavidin 1 showed 30%, 46%, 48%, and 49% homology to avidin, streptavidin, tamavidin 2, and tamavidin 1, respectively. Lentiavidin 2 showed 27%, 39%, 44%, 45%, and 85% homology to avidin, streptavidin, tamavidin 2, tamavidin 1, and lentiavidin 1, respectively. The cDNA encoding lentiavidin 1 is termed leav1 and that encoding lentiavidin 2 leav2. Nucleotide sequence data were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers AB675684 for leav1 and AB675685 for leav2. Primary structures of lentiavidins The 19 amino acids at the N-termini of lentiavidin 1 and lentiavidin 2 were characteristic of secretion signal peptides (Fig. 2), which are not present in tamavidins (10). The calculated molecular mass of the putative mature lentiavidin 1 and lentiavidin 2 was 14,415.4 Da and 14,403.6 Da, respectively. The calculated pI values were 3.9 and 4.4, respectively, which are very low (Table 1). Remarkably, lentiavidin 1 has the lowest in pI among the known biotinbinding proteins. It is, therefore, highly likely that the lentiavidins would show low levels of charge-driven non-specific binding to substances other than biotin. Eleven of 12 residues that directly associate with biotin in streptavidin were conserved in lentiavidin 1, and nine in lentiavidin 2 (Fig. 2), suggesting that lentiavidin 1 might have a high affinity for
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
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NOVEL BIOTIN-BINDING PROTEINS FROM LENTINULA EDODES
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FIG. 2. Amino acid sequences of biotin-binding proteins. The aligned sequences of avidin (GenBank accession number P02701), streptavidin (P22629), tamavidin 1 (AB102784), tamavidin 2 (AB102785), lentiavidin 1, and lentiavidin 2 are shown. Residues conserved across four or more of the sequences are shown in white type. Arrowheads indicate putative signal peptide cleavage sites. Asterisks indicate residues associated with biotin in streptavidin. The locations of primers are underlined.
biotin. One of the most important residues responsible for the tight biotin interaction in streptavidin is W120, which reaches from the neighboring subunit and helps to form a lid-like structure on top of the biotin-binding pocket, strengthening the binding affinity (24). Most of the tetrameric biotin-binding proteins studied to date possess the tryptophan residues analogous to W120 (W144 in Fig. 2) in streptavidin, such as W110 (W134 in Fig. 2) in avidin and W108 in tamavidin 2 (Fig. 2). In the lentiavidins, the residues at the corresponding positions are tryptophan (W136) in lentiavidin 1 and glycine (G136) in lentiavidin 2 (Fig. 2). Therefore, it is likely that
TABLE 1. Comparison of isoelectric points (pI) among biotin-binding proteins. Biotin-binding protein Vertebrate source Avidin AVR1, AVR6, AVR7 AVR2 AVR3 AVR4/5 Avm-pI4.7 Xenavidin Zebavidin Bacterial source Streptavidin Bradavidin Bradavidin II Rhizavidin Burkavidin Fungal source Tamavidin 1 Tamavidin 2 Tamavidin 2R104EK141E Lentiavidin 1 Lentiavidin 2
Origin
Gallus gallus G. gallus G. gallus G. gallus G. gallus Engineered protein Xenopus tropicalis Danio rerio
pI calculated 10.4 7.1 4.7 9.9 9.7 4.7 8.9 7.9
Streptomyces avidinii Bradyrhizobium japonicum B. japonicum Rhizobium etli Burkholderia pseudomallei
6.1 6.3
Pleurotus cornucopiae P. cornucopiae Engineered protein
6.2 7.4 5.1
Lentinula edodes L. edodes
3.9 4.4
pI measured
z7 z5 z10 z10 4.7
6.0 to 7.5
9.6 4.0 5.3
References
23 2 2 2 2 17 3 4 14 6 7 8 9
7.4 to 7.5 5.8 to 6.2
10 14 14 This study This study
lentiavidin 1 is a member of the avidin-like tetrameric biotinbinding proteins. Expression of lentiavidins in cell-free extracts and the biotin-binding activity Since lentiavidins were not expressed in E. coli (neither in the soluble form nor in the insoluble form), the insect cell-free expression system was employed to obtain active lentiavidins. The cDNA of lentiavidin 1 (leav1) or lentiavidin 2 (leav2), without the signal peptide, was cloned into an expression vector, and RNA transcribed from each cDNA was used for the protein synthesis in the cell-free extracts. The synthesized protein was then separated by non-denaturing SDS-PAGE, immobilized onto a membrane, and assayed for binding to biotinylated peroxidase. A band with a molecular mass of approximately 60 kDa was detected in the extracts containing the RNA from leav 1 (Fig. 3), but not in those from leav2 (data not shown). Thus, the immobilized lentiavidin 1 which was synthesized in the insect cell-free extracts bound to biotinylated peroxidase. Because the calculated molecular mass
1
2
(kDa) 75503725-
FIG. 3. Expression of lentiavidin 1 in insect cell-free extracts. Protein was synthesized with (lane 2) or without (lane 1) RNA from a coding sequence for lentiavidin 1 without a signal peptide, separated by non-denaturing SDS-PAGE, and transferred onto a PVDF membrane. Biotin-binding activity was then visualized.
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
TAKAKURA ET AL.
Fluorescence intensity (Photon counts)
4
50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0
0
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40
60
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The amount of cell-free extracts added (μl) FIG. 4. Binding of lentiavidin 1 with a fluorescent biotin. Biotin-4-fluorescein was incubated with cell-free extracts with (open squares) or without (closed squares) lentiavidin 1. Quenching of the fluorescence signal was then measured.
of the putative mature lentiavidin 1 monomer was 14,415.4 Da, the 60 kDa band was apparently a tetramer. Moreover, the free-form of the synthesized lentiavidin 1 was able to quench the fluorescence of biotin-4-fluorescein (Fig. 4), and the level of the quenching was proportional to the amount of extract assayed. Thus, lentiavidin 1 was clearly shown by two methods to have biotin-binding activity. Conclusion The present study led to the discovery of two novel avidin-like proteins, which we termed lentiavidins, in shiitake mushroom (L. edodes). The assay method used in this study will be useful for screening of avidin-like biotin-binding proteins from various organisms. L. edodes is the second fungus from which avidin-like proteins have been found, suggesting that avidin-like biotin-binding proteins might be distributed in the fungal kingdom more widely than anticipated. The function of lentiavidins in L. edodes remains unclear, but lentiavidin 1 may be involved in defense against various stresses, as has been suggested for other biotin-binding proteins. Avidin is induced by bacterial and viral infections and by cellular damage in chickens (25,26). Streptavidin was discovered as an antimicrobial agent in S. avidinii (27). Tamavidins also show antimicrobial activities against fungi (10,28), nematodes, insects, and amoebae (29). In our experimental condition, no active lentiavidin 2 was obtained. Since lentiavidin 2 does not have an essential residue for tight biotin-binding and inter-subunit associations, it might have a peculiar property. Although further studies are necessary for the efficient production of lentiavidins and for the detailed characterization of them, lentiavidin 1, with the lowest pI value of the known biotinbinding proteins, has a great potential to serve as a valuable tool with low non-specific binding in new applications and methodologies for avidin-biotin technology. ACKNOWLEDGMENTS The authors acknowledge Dr. Toshihiko Komari for his critical reading of the manuscript. References 1. Eakin, R. E., Snell, E. E., and Williams, R. J.: The concentration and assay of avidin, the injury-producing protein in raw egg white, J. Biol. Chem., 140, 535e543 (1941). 2. Laitinen, O. H., Hytönen, V. P., Ahlroth, M. K., Pentikainen, O. T., Gallagher, C., Nordlund, H. R., Ovod, V., Marttila, A. T., Porkka, E., Heino, S., and other 3 authors: Chicken avidin-related proteins show altered biotinbinding and physico-chemical properties as compared with avidin, Biochem. J., 363, 609e617 (2002). 3. Määttä, J. A., Helppolainen, S. H., Hytönen, V. P., Johnson, M. S., Kulomaa, M. S., Airenne, T. T., and Nordlund, H. R.: Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis, BMC Struct. Biol., 9, 63 (2009).
4. Taskinen, B., Zmurko, J., Ojanen, M., Kukkurainen, S., Parthiban, M., Määttä, J. A., Leppiniemi, J., Jänis, J., Parikka, M., Turpeinen, H., and other 6 authors: Zebavidin e an avidin-like protein from zebrafish, PLoS One, 8, e77207 (2013). 5. Tausig, F. and Wolf, F. J.: Streptavidin e a substance with avidin-like properties produced by microorganisms, Biochem. Biophys. Res. Commun., 14, 205e209 (1964). 6. Nordlund, H. R., Hytönen, V. P., Laitinen, O. H., and Kulomaa, M. S.: Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum, J. Biol. Chem., 280, 13250e13255 (2005). 7. Helppolainen, S. H., Määttä, J. A., Halling, K. K., Slotte, J. P., Hytönen, V. P., Jänis, J., Vainiotalo, P., Kulomaa, M. S., and Nordlund, H. R.: Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein, Biochim. Biophys. Acta, 1784, 1002e1010 (2008). 8. Helppolainen, S. H., Nurminen, K. P., Määttä, J. A., Halling, K. K., Slotte, J. P., Huhtala, T., Liimatainen, T., Ylä-Herttuala, S., Airenne, K. J., Närvänen, A., and other 5 authors: Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family, Biochem. J., 405, 397e405 (2007). 9. Sardo, A., Wohlschlager, T., Lo, C., Zoller, H., Ward, T. R., and Creus, M.: Burkavidin: a novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei, Protein Expr. Purif., 77, 131e139 (2011). 10. Takakura, Y., Tsunashima, M., Suzuki, J., Usami, S., Kakuta, Y., Okino, N., Ito, M., and Yamamoto, T.: Tamavidinsdnovel avidin-like biotin-binding proteins from the Tamogitake mushroom, FEBS J., 276, 1383e1397 (2009). 11. Green, N. M.: Avidin and streptavidin, Methods Enzymol., 184, 51e67 (1990). 12. Schetters, H.: Avidin and streptavidin in clinical diagnosis, Biomol. Eng., 16, 73e78 (1999). 13. Wilchek, M. and Bayer, E. A.: Introduction to avidin-biotin technology, Methods Enzymol., 184, 5e13 (1990). 14. Takakura, Y., Oka, N., Kajiwara, H., and Tsunashima, M.: Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties, J. Biosci. Bioeng., 114, 485e489 (2012). 15. Takakura, Y., Sofuku, K., and Tsunashima, M.: Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability, J. Biotechnol., 164, 19e25 (2013). 16. Takakura, Y., Suzuki, J., Oka, N., and Kakuta, Y.: Tamavidin 2-HOT, a highly thermo-stable biotin-binding protein with inter-subunit disulfide bridges, J. Biotechnol., 169, 1e8 (2014). 17. Marttila, A. T., Laitinen, O. H., Airenne, K. J., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S.: Recombinant NeutraLite avidin: a nonglycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties, FEBS Lett., 467, 31e36 (2000). 18. Alon, R., Bayer, E. A., and Wilchek, M.: Streptavidin contains an RYD sequence which mimics the RDG receptor domain of fibronectin, Biochem. Biophys. Res. Commun., 170, 1236e1241 (1990). 19. Takakura, Y. and Kuwata, S.: Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake, Biosci. Biotechnol. Biochem., 67, 2598e2607 (2003). 20. Sambrook, J. and Russell, D. W.: Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York (2001). 21. Kada, G., Falk, H., and Gruber, H. J.: Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate, Biochim. Biophys. Acta, 1427, 33e43 (1999). 22. Bayer, E. A., Ehrlich-Rogozinski, S., and Wilchek, M.: Sodium dodecyl sulfatepolyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin, Electrophoresis, 17, 1319e1324 (1996). 23. Marttila, A. T., Airenne, K. J., Laitinen, O. H., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S.: Engineering of chicken avidin: a progressive series of reduced charge mutants, FEBS Lett., 441, 313e317 (1998). 24. Sano, T. and Cantor, C. R.: Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin, Proc. Natl. Acad. Sci. USA, 92, 3180e3184 (1995). 25. Elo, H. A., Raisanen, S., and Tuohimaa, P. J.: Induction of an antimicrobial biotin-binding egg white protein (avidin) in chick tissues in septic Escherichia coli infection, Experientia, 36, 312e313 (1980). 26. Korpela, J., Kulomaa, M., Tuohimaa, P., and Vaheri, A.: Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage, EMBO J., 2, 1715e1719 (1983). 27. Chaiet, L., Miller, T. W., Tausig, F., and Wolf, F. J.: Antibiotic Msd-235. II. Separation and purification of synergistic component, Antimicrob. Agents Chemother. (Bethesda), 161, 28e32 (1963). 28. Takakura, Y., Oka, N., Suzuki, J., Tsukamoto, H., and Ishida, Y.: Intercellular production of tamavidin 1, a biotin-binding protein from Tamogitake mushroom, confers resistance to the blast fungus Magnaporthe oryzae in transgenic rice, Mol. Biotechnol., 51, 9e17 (2012). 29. Bleuler-Martinez, S., Schmieder, S., Aebi, M., and Künzler, M.: Biotin-binding proteins in the defense of mushrooms against predators and parasites, Appl. Environ. Microbiol., 78, 8485e8487 (2012).
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes) Yoshimitsu Takakura,1, *, x Kozue Sofuku,1 Masako Tsunashima,1 and Shigeru Kuwata2 Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan1 and Graduate School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan2 Received 21 April 2015; accepted 5 September 2015 Available online xxx
A biotin-binding protein with a low isoelectric point (pI), which minimizes electrostatic non-specific binding to substances other than biotin, is potentially valuable. To obtain such a protein, we screened hundreds of mushrooms, and detected strong biotin-binding activity in the fruit bodies of Lentinula edodes, shiitake mushroom. Two cDNAs, each encoding a protein of 152 amino acids, termed lentiavidin 1 and lentiavidin 2 were cloned from L. edodes. The proteins shared sequence identities of 27%e49% with other biotin-binding proteins, and many residues that directly associate with biotin in streptavidin were conserved in lentiavidins. The pI values of lentiavidin 1 and lentiavidin 2 were 3.9 and 4.4, respectively; the former is the lowest pI of the known biotin-binding proteins. Lentiavidin 1 was expressed as a tetrameric protein with a molecular mass of 60 kDa in an insect cell-free expression system and showed biotin-binding activity. Lentiavidin 1, with its pI of 3.9, has a potential for broad applications as a novel biotin-binding protein. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biotin-binding protein; Cell-free extract; Isoelectric point; Lentinula edodes; Non-specific binding]
Biotin-binding proteins have been found in a diverse array of organisms. For example, in vertebrates, avidin (1) and avidinrelated proteins (AVR1, AVR2, AVR3, AVR4/5, AVR6, AVR7) (2) were found in the chicken (Gallus gallus), xenavidin (3) in Xenopus tropicalis, and zebavidin (4) in Danio rerio. In bacteria, streptavidin (5) was found in Streptomyces avidinii, bradavidin (6) and bradavidin II (7) in Bradyrhizobium japonicum, rhizavidin (8) in Rhizobium etli, and burkavidin (9) in Burkholderia pseudomallei. In fungi, tamavidin 1 and tamavidin 2 (10) were found in tamogitake mushroom (Pleurotus cornucopiae). Avidin and streptavidin have been recognized for a long time and are well characterized. The dissociation constant (Kd) of biotin is 6 1016 M for avidin and 4 1014 M for streptavidin (11). The extraordinarily high affinities of the avidin- and streptavidin-biotin interactions are of great scientific value and have been exploited in diverse applications in medicine, biochemistry, and biotechnology (12,13). Among biotin-binding proteins, tamavidin 2 is unusual in that can be expressed at high levels in its soluble form in Escherichia coli, and it has a high affinity for biotin (10). Tamavidin 2 could, therefore, be produced industrially in E. coli and engineered to exhibit distinct characteristics such as a lower isoelectric point (14), reversible biotin-binding (15), or extremely high thermal stability (16). The detection or separation of biotinylated biomolecules from crude cell extracts is one of the most important applications of biotin-binding proteins, where the non-specific binding of proteins
* Corresponding author. Tel.: þ81 285 34 2655; fax: þ81 285 25 4460. E-mail address: [email protected] (Y. Takakura). x Present address: Leaf Tobacco Research Center, Japan Tobacco, Inc., 1900 Idei, Oyama, Tochigi 323-080, Japan.
to substances other than biotin can be extremely detrimental. In this regard, both avidin and streptavidin may show undesirable characteristics. Avidin, which is a basic glycoprotein, has shown a high level of non-specific binding to various biological components at physiological pH, resulting in high background levels (17). This high non-specific binding is thought to be due to its high isoelectric point (pI > 10). Although streptavidin has a lower pI (6.1e7.5), it contains an Arg-Tyr-Asp (RYD) tripeptide that apparently mimics the Arg-Gly-Asp (RGD) cell adhesion domain of fibronectin (18), thereby causing background binding in histochemical and cytochemical applications. The charge of a biotin-binding protein largely participates in non-specific binding to biological macromolecules. Since many biological macromolecules including DNA, RNA, acidic proteins or biomembranes are minus-charged in physiological pH, a biotinbinding protein with a lower pI value, which is minus-charged in such pH, may be effective to reduce the electrostatic chargedependent non-specific binding, and therefore be of value in various applications. Indeed, the mutant avidin Avm-pI4.7, with an acidic pI of 4.7, showed low non-specific binding to DNA and cells (17). A mutant tamavidin 2 with a pI of 5.8e6.2 also showed lower non-specific binding to DNA and glycoproteins from human sera compared with wild-type tamavidin 2 with a pI of 7.4e7.5 (14). Since mutations introduce the risk of disturbing the biotin-binding activity, an alternative approach could be to modify the proteins chemically or enzymatically, but such modification introduces the risk of creating heterogenous molecular species. Therefore, searching for novel biotin-binding proteins from unstudied sources has become an attractive approach. We screened various kinds of mushrooms for such proteins and here report the identification of
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Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
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two avidin-like proteins with pIs of 3.9 and 4.4, respectively in shiitake mushroom (Lentinula edodes).
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MATERIALS AND METHODS Extraction of proteins and nucleic acids Mushrooms including L. edodes and P. cornucopiae were purchased from local grocery stores. Total soluble proteins were extracted in 0.1 M HEPES-KOH pH 7.5, precipitated by using ammonium sulfate (75% saturate), then dissolved in and dialyzed against 20 mM HEPES pH 7.5 according to the procedure described by Takakura and Kuwata (19). Genomic DNA was isolated by the standard SDS-phenol method, and mRNA was purified according to the procedure described by Takakura and Kuwata (19). Biotin-binding assay The procedure for non-denaturing SDS-PAGE and staining of biotin-binding activity with biotinylated horseradish peroxidase (HRP) was described previously by Takakura et al. (16). Briefly, the protein sample was dissolved in 1 SDS gel-loading buffer (20) without reducing agent at room temperature for 10 min, separated by SDS-PAGE on a 10% gel, and then blotted onto a PVDF membrane (Millipore). The membrane was blocked with Trisbuffered saline (TBS) containing 3% BSA and 0.2% Tween 20, incubated in TBS containing 3% BSA with biotinylated HRP (Vector Laboratories) (1:1000 dilution) at room temperature for 1 h, and washed three times with TBS containing 0.2% Tween 20. The biotin-binding proteins were visualized by using ECL Detection Reagents (Amersham Biosciences). Streptavidin from S. avidinii (60 kDa as a tetramer, from Sigma) was used as a control. cDNA cloning PCR was performed with L. edodes genomic DNA as a template. The 50-ml reaction contained 100 ng of genomic DNA and 5 pmol of each degenerate primer in 1 GC buffer (TakaraBio). The forward primer was 50 -GGN ACI TGG TAT/C AAT/C G/CAA/G C/TTN GG-30 and the reverse primer was 50 -TAT/C TGN CCI G/CA/TC CAN GTN GT-30 , where N represents A, T, G, or C, and I represents inosine. Ex-taq (0.5 U) (TakaraBio) was incubated with Taq Start Antibody (Clontech) at room temperature for 10 min and then added to the reaction, which then underwent one cycle of 96 C for 3 min, 40 cycles of 96 C for 1 min, 50 C for 1 min and 72 C for 1 min, and one cycle of 72 C for 6 min. Amplified DNA was cloned into the pCR2.1Topo vector (Invitrogen) and sequenced. The PCR product was used as a hybridization probe to screen the L edodes cDNA library, which was constructed from 5 mg of mRNA by using a lZAP cDNA synthesis kit (Stratagene). cDNA clones were rescued as pBluescript plasmids from l phages by using in vivo excision according to the manufacturer’s instructions. DNA and amino acid sequences were analyzed by using GENETYX-WIN ver.8 software (Genetyx Co.). Cell-free protein synthesis The PCR product was amplified from each cDNA clone (leav 1 or leav2) by using the primers 50 -ATGGCTCCTACGACATTGACATCGAGGCAA-30 and 50 -TGATCAGGATCCCTACGCAATCTCAGCACGAG-30 for leav 1 or the primers 50 -ATGGCTCCTACGACATTGACATCAAGGCAA-30 and 50 -CAATGAGGATCCTTACGCAGTCTCAGCACGAA-30 for leav2. The termini of the products were filled in by using the Klenow fragment of DNA polymerase I (TakaraBio). Each product was then digested with BamHI and cloned into the expression vector pTD1 (Shimadzu) predigested with EcoRV and BamHI. RNA was synthesized from 10 mg of the linearized expression vector by using the T7 RiboMAX Express Large Scale RNA Production System (Promega) in a 200-ml reaction. Protein was synthesized from 1 mg of the RNA by using Transdirect insect cell (DTTþ) (Shimadzu) in a 1-ml reaction, which consisted of 500-ml of insect cell extract, 300-ml of reaction buffer, 20-ml of 4 mM methionine, 200-ml of RNA at 25 C for 5 h, according to the manufacturer’s instructions. A protein synthesis reaction without RNA was run as a negative control. A 50-ml scale synthesis was performed by using the FluoroTect GreenLys in vitro Translation Labeling System (Promega), and protein expression was verified by means of SDS-PAGE and fluorescence detection. Biotin-binding assay with fluorescent biotin Binding to biotin-4fluroescein was assayed according to the protocol described by Kada et al. (21) and Takakura et al. (10). Briefly, various quantities of the cell-free extracts were incubated with biotin-4-fluorescein (Molecular Probe, 25 pmol per 200-ml assay) at room temperature for 30 min. Fluorescence was measured by using an Infinite M200 microplate reader (Tecan).
RESULTS AND DISCUSSION Biotin-binding activity in L. edodes Hundreds of mushroom species were screened for biotin-binding activities, which were assayed after total soluble proteins were separated by nondenaturing SDS-PAGE and immobilized onto a membrane. This assay method is based on the characteristic of biotin-binding proteins that they retain their activities (16) as well as their oligomeric structures (22) in non-denaturing SDS-PAGE in which the heat-denature of protein sample is omitted. This characteristic seems to come from the property of biotin-binding
60 kDa
60 kDa
FIG. 1. Biotin-binding activity from mushrooms. Proteins from Lentinula edodes (Lane 2) and Pleurotus cornucopiae (Lane 1), and streptavidin (Lane 3) were separated on nondenaturing SDS-PAGE and transferred onto a PVDF membrane. Biotin-binding activity was then visualized by using a biotinylated peroxidase.
proteins that their inter-subunit associations are tolerant to SDS, but not to heat-denature. A clear signal of approximately 60 kDa was detected in the extract from L. edodes (Fig. 1), indicating that a biotin-binding protein was present in L. edodes. The size of the biotin-binding protein from L. edodes was similar to those of tamavidins from P. cornucopiae (10) and streptavidin. Some other mushrooms were also positive in the screen, but the signals were not as strong as that in L. edodes. We therefore focused on the protein from L. edodes in this study. Cloning of cDNAs for biotin-binding proteins from L. edodes Degenerate primers based on consensus sequences among the amino acid sequences of three biotin-binding proteinsdstreptavidin, tamavidin 1, and tamavidin 2dwere designed (Fig. 2). A DNA segment was amplified from the genomic DNA of L. edodes by PCR using these primers. The sequence was similar to those of known biotin-binding proteins. By using the PCR product as a probe, we obtained eight cDNA clones from the screen of 88,000 plaques in the L. edodes cDNA library. The eight cDNA clones were sequenced and classified into two types. The longest clones of both types encoded polypeptides consisting of 152 residues (Fig. 2). We termed the encoded proteins lentiavidins (L. edodes avidin). Lentiavidin 1 showed 30%, 46%, 48%, and 49% homology to avidin, streptavidin, tamavidin 2, and tamavidin 1, respectively. Lentiavidin 2 showed 27%, 39%, 44%, 45%, and 85% homology to avidin, streptavidin, tamavidin 2, tamavidin 1, and lentiavidin 1, respectively. The cDNA encoding lentiavidin 1 is termed leav1 and that encoding lentiavidin 2 leav2. Nucleotide sequence data were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers AB675684 for leav1 and AB675685 for leav2. Primary structures of lentiavidins The 19 amino acids at the N-termini of lentiavidin 1 and lentiavidin 2 were characteristic of secretion signal peptides (Fig. 2), which are not present in tamavidins (10). The calculated molecular mass of the putative mature lentiavidin 1 and lentiavidin 2 was 14,415.4 Da and 14,403.6 Da, respectively. The calculated pI values were 3.9 and 4.4, respectively, which are very low (Table 1). Remarkably, lentiavidin 1 has the lowest in pI among the known biotinbinding proteins. It is, therefore, highly likely that the lentiavidins would show low levels of charge-driven non-specific binding to substances other than biotin. Eleven of 12 residues that directly associate with biotin in streptavidin were conserved in lentiavidin 1, and nine in lentiavidin 2 (Fig. 2), suggesting that lentiavidin 1 might have a high affinity for
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
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FIG. 2. Amino acid sequences of biotin-binding proteins. The aligned sequences of avidin (GenBank accession number P02701), streptavidin (P22629), tamavidin 1 (AB102784), tamavidin 2 (AB102785), lentiavidin 1, and lentiavidin 2 are shown. Residues conserved across four or more of the sequences are shown in white type. Arrowheads indicate putative signal peptide cleavage sites. Asterisks indicate residues associated with biotin in streptavidin. The locations of primers are underlined.
biotin. One of the most important residues responsible for the tight biotin interaction in streptavidin is W120, which reaches from the neighboring subunit and helps to form a lid-like structure on top of the biotin-binding pocket, strengthening the binding affinity (24). Most of the tetrameric biotin-binding proteins studied to date possess the tryptophan residues analogous to W120 (W144 in Fig. 2) in streptavidin, such as W110 (W134 in Fig. 2) in avidin and W108 in tamavidin 2 (Fig. 2). In the lentiavidins, the residues at the corresponding positions are tryptophan (W136) in lentiavidin 1 and glycine (G136) in lentiavidin 2 (Fig. 2). Therefore, it is likely that
TABLE 1. Comparison of isoelectric points (pI) among biotin-binding proteins. Biotin-binding protein Vertebrate source Avidin AVR1, AVR6, AVR7 AVR2 AVR3 AVR4/5 Avm-pI4.7 Xenavidin Zebavidin Bacterial source Streptavidin Bradavidin Bradavidin II Rhizavidin Burkavidin Fungal source Tamavidin 1 Tamavidin 2 Tamavidin 2R104EK141E Lentiavidin 1 Lentiavidin 2
Origin
Gallus gallus G. gallus G. gallus G. gallus G. gallus Engineered protein Xenopus tropicalis Danio rerio
pI calculated 10.4 7.1 4.7 9.9 9.7 4.7 8.9 7.9
Streptomyces avidinii Bradyrhizobium japonicum B. japonicum Rhizobium etli Burkholderia pseudomallei
6.1 6.3
Pleurotus cornucopiae P. cornucopiae Engineered protein
6.2 7.4 5.1
Lentinula edodes L. edodes
3.9 4.4
pI measured
z7 z5 z10 z10 4.7
6.0 to 7.5
9.6 4.0 5.3
References
23 2 2 2 2 17 3 4 14 6 7 8 9
7.4 to 7.5 5.8 to 6.2
10 14 14 This study This study
lentiavidin 1 is a member of the avidin-like tetrameric biotinbinding proteins. Expression of lentiavidins in cell-free extracts and the biotin-binding activity Since lentiavidins were not expressed in E. coli (neither in the soluble form nor in the insoluble form), the insect cell-free expression system was employed to obtain active lentiavidins. The cDNA of lentiavidin 1 (leav1) or lentiavidin 2 (leav2), without the signal peptide, was cloned into an expression vector, and RNA transcribed from each cDNA was used for the protein synthesis in the cell-free extracts. The synthesized protein was then separated by non-denaturing SDS-PAGE, immobilized onto a membrane, and assayed for binding to biotinylated peroxidase. A band with a molecular mass of approximately 60 kDa was detected in the extracts containing the RNA from leav 1 (Fig. 3), but not in those from leav2 (data not shown). Thus, the immobilized lentiavidin 1 which was synthesized in the insect cell-free extracts bound to biotinylated peroxidase. Because the calculated molecular mass
1
2
(kDa) 75503725-
FIG. 3. Expression of lentiavidin 1 in insect cell-free extracts. Protein was synthesized with (lane 2) or without (lane 1) RNA from a coding sequence for lentiavidin 1 without a signal peptide, separated by non-denaturing SDS-PAGE, and transferred onto a PVDF membrane. Biotin-binding activity was then visualized.
Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003
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Fluorescence intensity (Photon counts)
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The amount of cell-free extracts added (μl) FIG. 4. Binding of lentiavidin 1 with a fluorescent biotin. Biotin-4-fluorescein was incubated with cell-free extracts with (open squares) or without (closed squares) lentiavidin 1. Quenching of the fluorescence signal was then measured.
of the putative mature lentiavidin 1 monomer was 14,415.4 Da, the 60 kDa band was apparently a tetramer. Moreover, the free-form of the synthesized lentiavidin 1 was able to quench the fluorescence of biotin-4-fluorescein (Fig. 4), and the level of the quenching was proportional to the amount of extract assayed. Thus, lentiavidin 1 was clearly shown by two methods to have biotin-binding activity. Conclusion The present study led to the discovery of two novel avidin-like proteins, which we termed lentiavidins, in shiitake mushroom (L. edodes). The assay method used in this study will be useful for screening of avidin-like biotin-binding proteins from various organisms. L. edodes is the second fungus from which avidin-like proteins have been found, suggesting that avidin-like biotin-binding proteins might be distributed in the fungal kingdom more widely than anticipated. The function of lentiavidins in L. edodes remains unclear, but lentiavidin 1 may be involved in defense against various stresses, as has been suggested for other biotin-binding proteins. Avidin is induced by bacterial and viral infections and by cellular damage in chickens (25,26). Streptavidin was discovered as an antimicrobial agent in S. avidinii (27). Tamavidins also show antimicrobial activities against fungi (10,28), nematodes, insects, and amoebae (29). In our experimental condition, no active lentiavidin 2 was obtained. Since lentiavidin 2 does not have an essential residue for tight biotin-binding and inter-subunit associations, it might have a peculiar property. Although further studies are necessary for the efficient production of lentiavidins and for the detailed characterization of them, lentiavidin 1, with the lowest pI value of the known biotinbinding proteins, has a great potential to serve as a valuable tool with low non-specific binding in new applications and methodologies for avidin-biotin technology. ACKNOWLEDGMENTS The authors acknowledge Dr. Toshihiko Komari for his critical reading of the manuscript. References 1. Eakin, R. E., Snell, E. E., and Williams, R. J.: The concentration and assay of avidin, the injury-producing protein in raw egg white, J. Biol. Chem., 140, 535e543 (1941). 2. Laitinen, O. H., Hytönen, V. P., Ahlroth, M. K., Pentikainen, O. T., Gallagher, C., Nordlund, H. R., Ovod, V., Marttila, A. T., Porkka, E., Heino, S., and other 3 authors: Chicken avidin-related proteins show altered biotinbinding and physico-chemical properties as compared with avidin, Biochem. J., 363, 609e617 (2002). 3. Määttä, J. A., Helppolainen, S. H., Hytönen, V. P., Johnson, M. S., Kulomaa, M. S., Airenne, T. T., and Nordlund, H. R.: Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis, BMC Struct. Biol., 9, 63 (2009).
4. Taskinen, B., Zmurko, J., Ojanen, M., Kukkurainen, S., Parthiban, M., Määttä, J. A., Leppiniemi, J., Jänis, J., Parikka, M., Turpeinen, H., and other 6 authors: Zebavidin e an avidin-like protein from zebrafish, PLoS One, 8, e77207 (2013). 5. Tausig, F. and Wolf, F. J.: Streptavidin e a substance with avidin-like properties produced by microorganisms, Biochem. Biophys. Res. Commun., 14, 205e209 (1964). 6. Nordlund, H. R., Hytönen, V. P., Laitinen, O. H., and Kulomaa, M. S.: Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum, J. Biol. Chem., 280, 13250e13255 (2005). 7. Helppolainen, S. H., Määttä, J. A., Halling, K. K., Slotte, J. P., Hytönen, V. P., Jänis, J., Vainiotalo, P., Kulomaa, M. S., and Nordlund, H. R.: Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein, Biochim. Biophys. Acta, 1784, 1002e1010 (2008). 8. Helppolainen, S. H., Nurminen, K. P., Määttä, J. A., Halling, K. K., Slotte, J. 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Takakura, Y., Oka, N., Kajiwara, H., and Tsunashima, M.: Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties, J. Biosci. Bioeng., 114, 485e489 (2012). 15. Takakura, Y., Sofuku, K., and Tsunashima, M.: Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability, J. Biotechnol., 164, 19e25 (2013). 16. Takakura, Y., Suzuki, J., Oka, N., and Kakuta, Y.: Tamavidin 2-HOT, a highly thermo-stable biotin-binding protein with inter-subunit disulfide bridges, J. Biotechnol., 169, 1e8 (2014). 17. Marttila, A. T., Laitinen, O. H., Airenne, K. J., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S.: Recombinant NeutraLite avidin: a nonglycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties, FEBS Lett., 467, 31e36 (2000). 18. Alon, R., Bayer, E. A., and Wilchek, M.: Streptavidin contains an RYD sequence which mimics the RDG receptor domain of fibronectin, Biochem. Biophys. Res. Commun., 170, 1236e1241 (1990). 19. Takakura, Y. and Kuwata, S.: Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake, Biosci. Biotechnol. Biochem., 67, 2598e2607 (2003). 20. Sambrook, J. and Russell, D. W.: Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York (2001). 21. Kada, G., Falk, H., and Gruber, H. J.: Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate, Biochim. Biophys. Acta, 1427, 33e43 (1999). 22. Bayer, E. A., Ehrlich-Rogozinski, S., and Wilchek, M.: Sodium dodecyl sulfatepolyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin, Electrophoresis, 17, 1319e1324 (1996). 23. Marttila, A. T., Airenne, K. J., Laitinen, O. H., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S.: Engineering of chicken avidin: a progressive series of reduced charge mutants, FEBS Lett., 441, 313e317 (1998). 24. Sano, T. and Cantor, C. R.: Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin, Proc. Natl. Acad. Sci. USA, 92, 3180e3184 (1995). 25. Elo, H. A., Raisanen, S., and Tuohimaa, P. J.: Induction of an antimicrobial biotin-binding egg white protein (avidin) in chick tissues in septic Escherichia coli infection, Experientia, 36, 312e313 (1980). 26. Korpela, J., Kulomaa, M., Tuohimaa, P., and Vaheri, A.: Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage, EMBO J., 2, 1715e1719 (1983). 27. Chaiet, L., Miller, T. W., Tausig, F., and Wolf, F. J.: Antibiotic Msd-235. II. 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Please cite this article in press as: Takakura, Y., et al., Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes), J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.003