Extremophiles (2014) 18:745–754 DOI 10.1007/s00792-014-0655-8

ORIGINAL PAPER

Self-assembled bionanoparticles based on the Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) coat protein as a prospective bioscaffold for nanotechnological applications Lei Song • Haina Wang • Shiwen Wang Hua Zhang • Haolong Cong • Li Huang Po Tien

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Received: 5 May 2014 / Accepted: 9 May 2014 / Published online: 6 June 2014 Ó Springer Japan 2014

Abstract Biomolecule-nanoparticle hybrid bioconjugates based on bioscaffolds such as protein cages and virus capsules have been widely studied. Highly stable and durable biotemplates are a vital pillar in constructing bio-inorganic functional hybrid composites. Here, we introduce a highly heat-resistant coat protein (CP) of Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) isolated from the hyperthermophilic archaeon as a prospective biological matrix. Our experiments showed that STSV1 CP was successfully cloned and solubly expressed in the Escherichia coli Rosetta-(DE3) host strain. Protein expression was verified by SDS-PAGE and western blot analysis of the reference C-terminally sixhistidine (His6) tagged STSV1 CP (HT-CP). Thermal stability experiments showed that the STSV1 coat protein Communicated by A. Driessen.

remained fairly stable at 80 °C. The proteins can be purified facilely by heat treatment followed by size exclusion chromatography (SEC). Transmission electron microscopy (TEM) analysis of the purified STSV1 CP protein aggregates demonstrated that the protein could self-assemble into rotavirus-like nanostructures devoid of genetic materials under our experimental conditions. Similar results were obtained for the HT-CP purified by heat treatment followed by Ni-NTA and SEC, indicating that moderately engineered STSV1 CP can retain its selfassembly property. In addition, the STSV1 CP has a high binding affinity for TiO2 nanoparticles. This illustrates that the STSV1 CP can be used as a bioscaffold in nanobiotechnological applications. Keywords Bioscaffold  STSV1 CP  Protein expression and purification  Protein nanoscale self-assembly  Affinity binding

Co-first authors: L. Song and H. Wang. L. Song  H. Zhang  H. Cong  P. Tien (&) CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China e-mail: [email protected] H. Wang  L. Huang (&) State Key Laboratory of Microbial Resources (SKLMR), Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China e-mail: [email protected] S. Wang National Center for Nanoscience and Technology, Beijing 100101, People’s Republic of China H. Zhang College of Life Science and Technology, HeiLongJiang BaYi Agricultural University, Daqing 163319, China

Introduction Biomolecules, particularly protein-derived nanoscaffolds, have many advantages over inorganic molecules in materials engineering and nanobiotechnological applications. Biomolecules allow for facile introduction and precise positioning of foreign ligands by genetic modification, easy preparation, self-assembling capabilities, startling uniformity, water solubility, low cytotoxicity and biocompatibility (MaHam et al. 2009; Uchida et al. 2007; Huang et al. 2005; Lee et al. 2009; Young et al. 2008). Based on bioscaffolds, many newly developed functional nanocomposites have exhibited novel applications in a variety of research fields ranging from functional materials to ingenious biomedical drug designs, including targeted delivery

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of nanomedicines (Smith et al. 2006; Garcea and Gissmann 2004), immunoassays (Sapsford et al. 2006), biomedical applications (Destito et al. 2009), bioimaging and biosensing (Medintz et al. 2005), nanoparticle (NP) synthesis via biomineralization (Knez et al. 2003; Mao et al. 2004; Nam et al. 2006) and energy conversion devices (Miller et al. 2007; Nuraje et al. 2012). In biotemplate-directed mineralization reactions, synthesis of well-defined NPs can be conducted in cage-like protein shells (Shenton et al. 2001) and spherical viruses (Douglas and Young 1998), and construction of bio-inorganic hybrid conjugates such as virus-quantum dot (QD) complexes by positioning the in situ participated nanocrystals on the pre-immobilized virus surface has been investigated (Lee et al. 2002). QDs with broader excitation wavelength ranges than those of conventional dyes and excellent photostabilities are used as novel fluorescent markers (Medintz et al. 2005; Bruchez et al. 1998). They can be used in high throughput biodetections and multicolor biomedical imaging when coupled with targeted biomolecules (Hu et al. 2006), as both quantum properties and biological functions are maintained. Stable interaction of QDs with biological matrix is vital in making hybrid conjugates, since the in situ crystallizing out of QDs from a precursor system is often performed under harsh conditions such as highly acidic, basic or thermal environments (Wang et al. 2005). Therefore, as the backbone of the conjugate, the bioscaffold must withstand harsh chemical conditions. TMV, as a remarkably stable virion that can endure high temperatures up to 60 °C and wide pH values ranging from 2 to 10 and hence remains as stable cylinders upon thermal and chemical treatments, has been widely studied in bioinorganic interactions (Knez et al. 2003; Shenton et al. 1999; Dujardin et al. 2003; Kobayashi et al. 2010). To cope with QDs manufacturing at even higher temperatures during the in situ fabrication of bio-inorganic nanoassemblies, for example, the ZnO QDs preparation at temperature as high as 80 °C (Fu et al. 2007), we have focused on the selfassembling behavior and mineral-binding affinity of the coat protein of STSV1, a spindle-shaped virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis isolated from a field sample from Tengchong in China (Xiang et al. 2005). Extremophiles have adapted to living in extraordinarily hot, highly acidic or salty environments. It has been shown that the STSV1 virion consists primarily of a single coat protein, encoded by ORF 40, with a calculated molecular mass weight of 15.6 kDa and an isoelectric point of 9.9, which presumably plays a key architectural part in the assembly of the nucleocapsid of STSV1. Given the high temperature of above 80 °C of STSV1 habitat, it is reasonably predicted that STSV1 CP is an ideal heat-resistant biotemplate. Here, we report that the STSV1 CP gene was

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successfully cloned and efficiently expressed in the E. coli gene expression system, and that the recombinant coat proteins were purified readily by heat treatment followed by size exclusion chromatography (SEC) separation. TEM observation of the protein aggregates revealed the formation of ring-like viroid structure through self-assembly of the STSV1 CP. In addition, adsorption experiments showed that the STSV1 CP had a higher avidity for TiO2 nanoparticles. As far as we know, this is the first report on the in vitro self-assembling property and binding affinity of the STSV1 CP. Aimed at NPs application in life sciences, our preliminary work suggests that the highly heat-resistant STSV1 CP may be developed into a prospective biological matrix for construction of compound structures for nanotechnological use.

Materials and methods Construction of expression plasmids for STSV1 wildtype CP (WT-CP) and His-tagged CP (HT-CP) The plasmid containing the STSV1 WT-CP gene was constructed as follows. First, the WT-CP gene was PCR amplified from the STSV1 genomic DNA using the forward primer 50 -CATCATCCATATGGCAAGAGAGGAA CCATACAAG-30 and the reverse primer 50 -ATCGCA CTCGAGTTACATACTAGCCTGCATAGTTA-30 (with the restriction sites of NdeI and XhoI underlined). The PCR products were then digested with NdeI and XhoI, and ligated into the appropriately cleaved pET-30a expression vector to construct the recombinant pET-WCP plasmid for WT-CP overproduction. By replacing the above reverse primer with the following primer 50 -GCACTCGAGTTAGTGATGGTG ATGGTGATGCATACTAGCCTG-30 (with a XhoI site underlined and six-histidine encoding sequence in italics), the plasmid pET-HCP for the overproduction of His-tagged STSV1 CP (HT-CP) was prepared. Overproduction of recombinant STSV1 coat proteins in bacteria The recombinant plasmids pET-WCP and pET-HCP were verified by sequencing analysis, followed by transformation into the E. coli Rosetta-(DE3) competent cells. After incubation expression for 3 h or indicated lengths of time under the conditions of induction with isopropyl thiogalactopyranoside (IPTG) (1 mM), the cell pellets were collected from the cultures by centrifugation at 5,000 rpm and resuspended in phosphate-buffered saline (PBS, pH 7.4). The cells were lysed by sonication and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants were harvested and stored at 4 °C.

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SDS-PAGE and western blot analysis of recombinant proteins To identify the soluble expression of the recombinant proteins, IPTG-induced and -uninduced culture-derived supernatant lysate samples as well as the pET-WCP-induced whole cells were prepared for SDS-PAGE, and the supernatant sample from pET-HCP-induced cell lysates was subjected to western blot. In brief, proteins were solubilized in SDS-PAGE lysis buffer, boiled at 100 °C for 5 min and then run on a 15 % polyacrylamide gel. For the western blot experiment, proteins separated by SDS-PAGE were transferred to a PVDF membrane. The membrane was then blocked with 5 % milk in TBS for 30 min at 37 °C and incubated with mouse monoclonal anti-His antibody (primary antibody, diluted 1:200) at 4 °C overnight. After three washes with TTBS (TBS containing 0.625 % Tween 20), the membrane was incubated with HRPconjugated goat anti-mouse IgG secondary antibodies (diluted 1:2000) for 1 h at 37 °C. After another three washes in TTBS, the blots were developed with a Chemiluminescence Detection Kit for HRP. Thermal stability of recombinant STSV1 WT-CP Heat-resistant capability of the recombinant STSV1 WT-CP was investigated by examining the variation of the protein content in the SDS-PAGE analysis. Equal amounts of the stored supernatant WT-CP protein were fractionated into a series of sample tubes, and the samples were incubated at temperatures ranging from 65 to 85 °C. After 30 min, each sample was centrifuged at 12,000 rpm. The supernatants were subjected to SDS-PAGE on a 15 % polyacrylamide gel. Purification of STSV1 WT-CP and HT-CP In view of the heat-resistance of the recombinant proteins, the facile purification procedure was performed. First, the stored WT-CP and HT-CP cell extracts were exposed to heating at 80 °C for 1 h, followed by centrifugation at 12,000 rpm for 30 min to enable the thermal-denatured proteins to pellet. The supernatants were then dialyzed in buffer (20 mM Tris, 50 mM NaCl, 0.5 % DMSO, 5 % glycerin, pH = 7.0), and further purified by SEC on a Sephadex G75 column using the AKTA Explorer FPLC system (Amersham Pharmacia Biotech, Inc., USA) with the above buffer as the mobile phase and at a flow rate of 1.0 ml min-1. Finally, purified WT-CP and HT-CP proteins eluted as aggregates on the SEC column were collected. Self-assembly assays of recombinant STSV1 WT-CP The WT-CP protein aggregate sample was prepared for TEM observation. A 20-ll protein sample solution was

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placed on carbon-coated 200 mesh copper grid for 1 min and then negatively stained with 2 % uranyl acetate. After the specimen grid was dried under an infrared lamp, TEM images were recorded on a JEM-1400 electron microscope operating at 80 kV. TEM observation of the self-assembly of HT-CP and immunogold labeling TEM analysis The purified HT-CP cell lysates were further subjected to Ni-NTA column affinity chromatography for further purification. According to the ProBond Purification protocol, samples were loaded on HisTrap HP columns (pre-charged with Ni2?, GE Healthcare) pre-equilibrated with 4 column volumes of binding buffer A (20 mM Tris–HCl, pH = 8.0; 50 mM NaCl), followed by washing with 4 column volumes of buffer A to remove non-specific binding proteins. The bound proteins were eluted with a linear concentration gradient of imidazole in buffer A. Fractions were collected in 1 ml aliquots and examined by SDS-PAGE analysis. The HT-CP-rich fractions were pooled and dialyzed in buffer A, further purified by SEC again as above with buffer A as the mobile phase. The purified HT-CP aggregate sample was subjected to TEM analysis as above. Immunogold labeling in TEM analysis was conducted to identify the binding function of the hexahistidine group in HT-CP-based structures. Carbon-coated grids were floated on a drop (20 ll) of purified HT-CP VLP aggregates for 1 min, then blocked with 3 % BSA for 5 min and then exposed to mouse anti-His monoclonal antibody (MAb) (1:20 dilution in 3 % BSA) for 15 min. The grids were washed three times with PBS containing 0.05 % Tween 20 for 3 min each, and then exposed to anti-mouse IgG antibody conjugated with 5 nm gold particles (1:10 dilution in 3 % BSA) for 15 min. After three washes with PBS and three more washes with MilliQ water, the grids were negatively stained with 1 % uranyl acetate and then examined at 80,0009 magnification with a JEM 1400 electron microscope. Affinity adsorption of recombinant STSV1 CPs to NPs An absorption experiment was performed to investigate the affinity binding capability of recombinant STSV1 CPs for certain NPs. The affinity and specificity of binding of STSV1 CP aggregate samples with widely studied TiO2 nanopowders (Degussa TiO2 P25) and ZnO NPs (presented by Dr. Peng Hu from IPE-CAS in China, see reference (Fangli et al. 2003) were investigated. In the binding experiment, about 0.5 mg of each NP was mixed homogeneously in 1-ml distilled water by sonication for 5 min. Then, 20 ll of each NP suspension was added to 500 ll of the WT-CP protein aggregate solution, and the mixture was

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Fig. 1 a SDS-PAGE analysis of recombinant WT-CP and HT-CP (right panel) and western blot identification of recombinant HT-CP (left panel). Protein samples loaded onto the polyacrylamide gel were: uninduced WT-CP strain, induced WT-CP whole cell lysate, induced WT-CP supernatant after sonication of whole cells, uninduced HT-CP strain and induced HT-CP supernatant (lane 1, 2, 3, 4 and 5,

respectively). Lane M, molecular weight markers. The WT-CP and HT-CP protein bands migrating at *15 and 17 kDa MW, respectively, are indicated by an arrow. b Induction time course. Protein samples were induced with IPTG for 3/4/5 h (lane a, b, c, respectively) and the supernatant (lane d). Lane M, molecular weight markers

incubated with gentle agitation for 3 h in a 4 °C laminar flow hood to allow the components to bind to each other. Afterwards, the two mixtures were, respectively, centrifuged to get precipitant and supernatant samples. In addition, the HT-CP’s binding affinity for TiO2 NPs was compared with that of WT-CP to examine the influence of C-terminally fused foreign peptide of His6 tag. Samples of the two protein aggregates were each mixed with the target NPs in different proportions at 4 °C for 1 h with gentle agitation. More TiO2 NPs were used for binding with respect to HT-CP. For these experiments, changes in the protein contents between before and after adsorption were visually examined in SDS-PAGE gels.

panel). Western blot analysis revealed that the recombinant HT-CP protein could be specifically recognized by mouse anti-His antibody (Fig. 1a, left panel). In addition, an induction time course experiment (as shown in Fig. 1b) indicated that, with increasing induction time from 3 to 5 h, the expressed protein content no longer increased.

Results Construction of recombinant plasmids and expression of WT-CP and HT-CP The two recombinant plasmids of pET-WCP and pET-HCP for the expression of recombinant WT-CP and HT-CP, respectively, were successfully constructed (see ‘‘Materials and methods’’), and the insert genes were all confirmed by sequencing analysis. After transformation of the two plasmids into the E. coli Rosetta-(DE3) competent cells and induction by IPTG, the recombinant expression of WT-CP and HT-CP was identified by SDS-PAGE analysis. The results showed that, as compared to their respective uninduced cell culture sample, the overexpressed soluble WTCP and HT-CP proteins were banded clearly on the polyacrylamide gel with the expected molecular weights (MW) of about 15 kDa and 17 kDa, respectively (Fig. 1a, right

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Thermal stability of recombinant STSV1 WT-CP When the STSV1 WT-CP lysate was subjected to heat treatment at different temperatures ranging from 65 to 85 °C, white floccular insoluble denatured proteins appeared in each protein sample. After centrifugation, SDS-PAGE of each supernatant sample was conducted. We examined the variation of the remaining target protein content in each supernatant sample upon heat treatment. As seen from Fig. 2, with increasing temperature, the target protein in the WT-CP supernatant sample became increasingly pure, as the other proteins decreased due to heat-triggered denaturation. Although the amount of target protein appeared to decrease slightly when the temperature exceeded 70 °C, comparatively large amounts of the protein still existed steadily until 82 °C. It is concluded that STSV1 WT-CP could exhibit superior thermal stability at temperatures as high as 80 °C. The HT-CP’s thermal stability was similarly verified (data not shown). Purification of recombinant STSV1 CPs According to ‘‘Materials and methods’’, STSV1 WT-CP was first exposed to heat treatment to remove denatured proteins, and then subjected to SEC separation. The result of the chromatographic analysis was shown in Fig. 3. The

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Fig. 2 Thermal stability of recombinant WT-CP at different temperatures ranging from 65 to 85 °C

highest chromatographic absorption peak (95 % of the total eluted proteins, marked as 1 in Fig. 3) contains WT-CP protein aggregates, suggesting a relatively higher purity of the protein. In addition to the negligible absorption peaks comprising of very small amounts of other proteins, the WT-CP monomer absorption peak was obtained (peak 2 in Fig. 3). The identity of WT-CP protein aggregates and monomers was determined by SDS-PAGE analysis, in which a single specific protein band positioned at about 15 kDa matched perfectly with the MW of WT-CP (data not shown). Using the same procedure as above, HT-CP protein was purified in a similar manner. The two purified proteins were stored at 4 °C. Self-assembly of recombinant STSV1 WT-CP protein The purified WT-CP protein corresponding to number 1 peak was prepared for TEM examination. Direct observation of negatively stained sample grids by TEM showed that the protein could self-assemble into regularly formed

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protein nanoassemblies (PNs) (Fig. 4). Many scatteredly distributed circular PNs were seen in a large visual field (Fig. 4a). The uniformly formed rotavirus-like nanostructures (Fig. 4b) were dominant. Large protein nanoparticles of *80 nm in diameter were observed (Fig. 4c). Isolated PNs were predominantly observed, however, there existed as well the circumstance of PNs cluster, with a few WT-CP self-assembled PNs gathering into an agglomerate which could be visualized in Fig. 4d. The TEM image of a single WT-CP nanoassembly structure showed clearly that the protein nanoparticle appeared to be constituted by a sharp ring-like solid peripheral boundary encompassing a hollow center area (Fig. 4e, f). This was more apparent in the large particle of about 80 nm in diameter; the circular boundary was 8 nm and the center hollow. The detailed assembly characteristics of the WT-CP protein are currently being investigated. TEM observation of self-assembly of HT-CP and immobilization of immunogold NPs on HT-CP PNs SDS-PAGE analysis of the HT-CP isolated by Ni–NTA showed that the addition of a His tag facilitated the further purification of HT-CP. Elution buffers containing 50, 100 and 200 mM imidazole were used to optimize HT-CP purification conditions. The results indicated that most of the non-HT-CP proteins were eluted in the 100-mM imidazole fraction, and the pure HT-CP was harvested in the 200-mM imidazole eluate, with a single band seen on the SDS-PAGE gel (data not shown). After dialysis against buffer A and subsequent purification by SEC, the purified HT-CP aggregate was subjected to TEM analysis. The TEM analysis showed that HT-CP could assemble into

Fig. 3 Purification of STSV1 WT-CP protein aggregates by SEC. Aggregate peak marked with number 1 and monomer absorption peak numbered by 2, respectively, were noted in the chromatographic diagram

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Fig. 4 TEM analysis of the self-assembly of the purified WT-CP. a Dotted-star-like WT-CP protein nanoassemblies visualized in a wider field of vision. b Rotavirus-like nanostructures with the uniform

size. c Larger protein nanoparticle. d Agglomeration of WT-CP PNs. e Clear image of a single PN. f Peripheral boundary and empty center area in a larger single PN. Bars are indicated

circular PNs just as WT-CP (Fig. 5). Some single-dispersed circular HT-CP PNs could also be seen (Fig. 5a). The uniformly formed rotavirus-like nanostructures indicated by Fig. 5b are clearly seen. A TEM image of a single HT-CP nanoassembly structure also showed that the protein nanoparticles consist of a ring-like boundary and a hollow center (Fig. 5c). The binding ability of the His tag in HT-CP was tested by immunogold labeling in TEM. The existence of the His tag was first confirmed by western blot analysis. Results from the immunogold TEM analysis showed that when the HT-CP aggregates were labeled with mouse anti-His primary mAb followed by incubation with 10-nm gold-conjugated anti-mouse IgG secondary antibody, the immobilization of gold NPs on the non-sharp ring-like HTCP assembled structures was seen (Fig. 6). Repeated drying and washing with the elution detergents during TEM sample preparation may cause a little distortion of the protein assemblies. Moreover, it can be seen that the HTCP PNs can form agglomerated clusters. As expected, very few gold nanoparticles could be found on the control Cu TEM grid (data not shown). It can be concluded that

C-terminally fused His tag does not interfere with STSV1 CP’s self-assembly property.

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Affinity adsorption of recombinant STSV1 CPs to TiO2 NPs The binding affinity of the recombinant STSV1 WT-CP for extensively researched TiO2 NPs and its interaction with inorganic particles were examined in an absorption comparison experiment with the equally well-studied ZnO NPs as a control. SDS-PAGE analysis (Fig. 7) showed that large quantities of WT-CPs, which migrated as a specific band positioned at the expected molecular weight of *15 kDa (indicated by arrow in the gel), were sedimented upon incubation with TiO2 NPs, whereas only a small amount of WT-CPs were precipitated after incubation with ZnO NPs. In addition, it was unexpected that, although three times as much TiO2 NPs as that used for binding with WT-CP were incubated with purified HT-CP lysates, the amount of precipitated HT-CPs appeared at a relatively low level. These results indicate that the recombinant STSV1 WT-CP has a strong affinity for TiO2 NPs.

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Fig. 5 TEM analysis of the self-assembly of the purified HT-CP. a HT-CP protein nanoassemblies (indicated by arrow) visualized in a wider field of vision. b Rotavirus-like nanostructures with the uniform size (indicated by arrow). c Clear image of a single HT-CP PN. Bars are indicated

Fig. 6 Electron micrograph showing immunogold labeling of His tag in the HT-CP aggregate sample. The black dots of gold nanoparticles attaching to the Cu TEM grid indicate the presence of His-tags on the protein nanoassemblies. Bar 100 nm

Discussion Robust virus coat protein-derived bionanoparticles are an ideal scaffold for constructing hybrid functional materials. They offer biocompatibility, bio-safety (genome-free, noninfectious and non-replicating properties) and high stability. The coat protein components of viruses infecting extremophiles presumably are very stable to the harsh conditions, making them valuable in bionanoparticle development. In this work, we report the potentialities of the coat protein of STSV1, which infects a hyperthermophilic archaeon as the self-assembled bioscaffold. We developed an E. coli-based system to efficiently express the recombinant STSV1 CPs. The design of HT-CP construct would enable the readily purification of the recombinant HT-CP by Ni–NTA affinity chromatography

Fig. 7 SDS-PAGE analysis of protein lysate samples in an adsorption comparison experiment. The experiment identified the binding affinity of the recombinant WT-CP for TiO2 NPs by examining variation in protein content after incubation with target NPs against the control systems. Protein samples loaded onto 0.1 % SDS-15 % polyacrylamide gels (from left to right) were: protein markers (lane M); untreated WT-CP lysate before binding; pellet (P) from WT-CP lysate incubated with TiO2 NPs and centrifuged; supernatant (S) from WT-CP lysate incubated with TiO2 NPs and centrifuged; pellet (P) from WT-CP incubated with ZnO NPs and centrifuged; supernatant (S) from WT-CP incubated with ZnO NPs and centrifuged; pellet (P) from untreated WT-CP lysate after binding as blank control; untreated HT-CP lysate; pellet (P) from HT-CP lysate incubated with TiO2 NPs and centrifuged. Large amounts of sedimented WT-CP proteins were indicated by arrow. S represents abbreviation for supernatant and P for pellet

and allows for investigation of the effect of foreign peptide fused at the C-terminus of the protein. It should be noted that the expression of recombinant proteins failed in the E. coli BL 21 series of expression host strains. The reason should be attributed to a high abundance of rare codons existing in the STSV1 CP gene where, as counted, six codons among 7 rare codons, including AGA, AGG, AUA,

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CUA, GGA, and CCC, were found. Archaea are phylogenetically distinct from Bacteria and Eukaryotes (Koonin et al. 1997). So, archaeal virus may have codons rarely used in E. coli, which would affect the expression of the archaeal protein in the bacterium. When using the Rosetta(DE3) host strain with a compatible chloramphenicolresistant plasmid supplying tRNAs for 7 rare codons in our experiment, successful expression of recombinant STSV1 CPs was achieved. The result from the experiments showed that both STSV1 WT-CP and HT-CP demonstrated high heat-resistance capabilities. Because the host strain of STSV1 has an optimum growth temperature above 80 °C, it was reasonably predicted that virus coat protein might remain stable at high temperature of up to 80 °C. The experiment results have proved this prediction. Moreover, this thermal stability remained unaffected when the His6 tag was included at the C-terminus of WT-CP, which would be a reference for incorporating the foreign functional peptide into STSV1 CP for the hybrid structure design. We suggest that the superior thermal stability of the STSV1 CP is related to its structure, and certain amino acids may play a pivotal role in maintaining the protein’s specific spatial conformation. Analysis of its amino acid sequence using the EditSeq computer software (DNAStar) shows that the STSV1 CP contains 17 strongly basic amino acids (R and K), 36 polar amino acids and 53 hydrophobic amino acids among a total of 144 aa. As we know, hydrophobic interaction is endothermic and entropy-driven reaction, which stabilizes protein at a high temperature. So we speculate that the high content of hydrophobic amino acids would greatly contribute to the heat resistance of STSV1 CP. An understanding of the crystal structure of STSV1 CP and the detailed heat-resistance mechanism need to be elucidated in future work. The exact mechanism of the recombinant STSV1 WTCP’s ability to self-assemble into rotavirus-like bionanoparticles remains unknown. As reported before, it appears to be an intrinsic property for the viral CP to form a viruslike particle (VLP) (Kegel and van der Schoot 2006). For example, Tobacco Mosaic Virus (TMV) CP can form several types of aggregates in solution, such as monomers, oligomers, disc-like assembly and helices interconverting reversibly upon specific variations of temperature, pH and ionic strength (Durham et al. 1971; Kegel and van der Schoot 2006). Similarly, protein interactions including hydrophobic interactions, electrostatic interactions and the formation of ‘Caspar’ carboxylate pairs may account for the assembly process of STSV1 CP. In addition, many important modeling and kinetic studies concerning virus assembly were based on the assumption that assembly is driven towards the lowest free energy state (Ding et al. 2010; Nguyen et al. 2009). In light of this, ring-like

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nanostructures should be the most stable form under our experimental conditions. In addition to the predominant PN of about 50 nm in diameter, a few larger PNs sized about 80 nm were formed as well, which may be attributed to small discrepancies in the local chemical microenvironments. There existed the possibility of shrinking and swelling in the assembled capsid shells as a function of the pH value and water contents. Non-homogeneity of sample solution and existence of the impurities in a certain area might randomly result in the variability of PNs. Besides, there may be individual distortions of protein assembly structures during the TEM sample preparation and negative stain process. In our opinion, STSV1 WT-CP would normally assemble into the uniform-sized ring-like PNs of about 50 nm in diameter, as was also found in the HT-CP self-assembly test. Furthermore, the better dispersion of PNs can be seen in TEM observation, probably because of the electrostatic repulsion of the charged protein molecules. In addition, the depolymerizing agents such as DMSO and glycerin in the buffer used in SEC might play a promoting role in retarding the PNs’ agglomeration. However, a few clusters containing several STSV1 WT-CP PNs were also found. The reason was probably that the assembled PNs themselves were susceptible to aggregation under certain conditions, and the electrostatic screening function caused by local salty microenvironment can also promote the evolution of aggregation. With respect to the HT-CP self-assembly, it was probably the existence of His tag that mainly contributed to the agglomeration of the protein nanoassemblies seen in immunogold labeling TEM photograph via p-stacking mechanisms (Bruckman et al. 2011). Significantly, the fusion of the His tag had no interference with the STSV1 CP’s self-assembly property, which could be very valuable and extended to other functional peptides of interest for wide-spread applications in nanobiotechnology. That HT-CP retained the same selfassembly behavior as that of wild-type STSV1 CP suggests that the His tag was probably inserted in a permissive site of the carboxyl terminus of STSV1 CP, causing no interference in the protein’s natural function and no steric hindering of the dynamics of the assembly process. To identify more workable insertions requires a deep understanding of the structure and assembly properties of STSV1 CP. Other conditions suitable for STSV1 WT-CP and HTCP self-assembling into regular structures still need to be explored; and the detailed mechanism of self-assembly needs to be further investigated. As we know, through evolution nature has invented many biomolecules which can interact well with the inorganics and have wonderful biomineralization ability (Sarikaya et al. 2003). In biohydrometallurgy research field, many thermophilic bioleaching microorganisms show a specific absorption with certain minerals (Mikkelsen et al.

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2007). Hot springs are rich in a variety of minerals coming out from underground through volcanic vents. It is speculated that our STSV1 WT-CP products might have better interactions with certain minerals. Currently, TiO2 NPs as the optimal semiconductive photocatalyst have been widely developed for various applications in industrial and medical fields due to their superior properties such as nontoxicity, low cost, high activity and strong stability (Yin et al. 2013; Xu et al. 2007). It has been an intriguing subject in bio-inorganic hybrid conjugates research (Jolley et al. 2011). There is also a great interest in ZnO nanoparticles nowadays because of their uniquely optical and electrical properties which found applications in organic– inorganic interactions studies (Tomczak et al. 2009; Song et al. 2010). The two commonly used NPs were selected for exploration of their interactions with the STSV1 WT-CP. Unexpectedly, in our comparison experiments on the binding ability, the STSV1 WT-CP had a more efficient binding with TiO2 NPs, as compared to that with ZnO NPs. Previous studies (Sarikaya et al. 2003; Li and Kaplan 2003) have reported that the surface-active functional groups on a protein, such as basic amino acids (arginine and lysine) and hydroxyl-containing residues common in metal oxidebinding peptides, may interact with minerals via coordination with metal ions, through hydrogen bonding, polarity, charge effects and structural recognition mechanisms. According to EditSeq analysis, a relatively higher proportion of R and K as well as polar amino acids would confer STSV1 WT-CP the binding affinity for the metal oxides. Moreover, a certain specific TiO2 NPs-binding motif in the STSV1 WT-CP, like a hexapeptide previously reported in the literature (Sano and Shiba 2003), might promote the bio-inorganic interaction. Structural analysis of STSV1 CP would elucidate more detailed molecular mechanisms responsible for its binding ability. We infer that the weak HT-CP binding affinity for TiO2 NPs is because the 6-histidine tag in the C-terminus might cause the side-byside aggregation of the proteins by p-stacking mechanisms (Bruckman et al. 2011), and hence enhance agglomeration of the HT-CP PNs by overcoming the electrostatic repulsion between neighboring PN subunits, which would increase the steric hindrance of the binding process. There also exists the possibility of conformational changes which would shield the active motif. The exact mechanism between the protein and NPs awaits further investigation. In conclusion, we have introduced the self-assembling property and binding ability of a highly thermal-stable protein-STSV1 CP. Our work may be helpful in dealing with some limitations in fabricating functional bio-inorganic hybrid nanoarchitectures under critical reactive conditions. As STSV1 WT-CP VLP interacts well with TiO2 NPs, there are exciting potential applications such as new nanoplatforms for use in bioremediation of heavy

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metal pollutants, and novel bio-nanocomposites that can be explored. This bionanoparticle template can be readily modified by genetic recombination, for example, genetic fusion with inorganic binding peptide for minerals of interest. The structural analysis of the STSV1 CP should be investigated for future genetic engineering design. In addition, immunogenicity of STSV1 CP VLP matrix should be investigated to assure its biocompatibility for use in biomedical therapy. With the increasing study on the applications of extremophiles (Norris et al. 2000; Egorova and Antranikian 2005) and rapid development of bionanotechnology, many NPs in combination with durable nanoscale biotemplates can be used in biomedical and industrial fields, such as quantum dot-based biotracking and bioimaging use in biodetection and energy conversion device design. This work may be helpful in developing a generic approach for bio-inorganic engineering based on hyperthermophilic PNs. Acknowledgments We are grateful to Prof George F. Gao’s research group from IM, CAS for generous support. The authors wish to thank Ms. Jingnan Liang from IM, CAS for TEM analyses. We also thank Prof. Paul Chu, guest professor of Institute of Microbiology, Chinese Academy of Sciences, for critical reading of the manuscript and providing constructive comments and suggestions. This work was supported by Grants from the National Basic Research Program of China (973 Program, Grant Nos. 2011CB504703 and 2010CB530102) and the National Natural Science Foundation of China (NSFC, Grant Nos. 81321063 and 31270211).

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