Characterization and expression of a protein involved in gold nanoparticle formations by Thermus scotoductus SA-01 Mariana Erasmus a, *, Jacobus Albertyn a , José Berenguer b , Esta van Heerden a a UFS/BioPAD Metagenomics Platform, Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, 9301, South Africa. b Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049, Spain. *Tel: +27 51 401 3391, Fax: +27 51 444 3219, E-mail: erasm@ufs.ac.za Introduction Gold nanoparticles have a wide variety of uses, as the material properties at nanoscale differ from that of the bulk form. These differences can be due to an increase in relative surface area and an increase in the dominance of quantum effects. These changes then influence the optical, magnetic and electronic properties of the material and results in the application of gold in diverse areas, such as catalysis, biolabeling, nonlinear optical devices and optical recording media, colours and coatings, as well as the biomedical sector. Micro-organisms such as fungi have been found to produce nanoparticles of different shapes and sizes with better monodispersity than when using chemical methods, and the microbial interaction with metals will also supply more eco-friendly methods for nanoparticle production. For tailor-made nanoparticles synthesis a possibility of optimizing specific size and shape of nanoparticles has been demonstrated. Temperature, pH and other conditions were varied and specific shapes of gold nanoparticles were obtained. Thermus scotoductus SA-01, a thermophilic bacterium, isolated from a deep gold mine, was used for purification of a gold(III) reducing and novel nanoparticle synthesizing protein. This discovery shows that gold nanoparticles can be produced by a peptide-binding protein. The interaction of the protein extracted and purified from Thermus scotoductus SA-01, as well as the recombinant proteins, with Au 3+ under varying physico-chemical conditions have been studied using TEM, EDX, and by measuring the surface plasmon resonance band, to illustrate the effect on particle morphology and to elucidate the protein mechanism. Expression Characterization Mukherjee and co-workers found that gold nanoparticles exhibit pink-purple colours, which arises due to excitation of surface plasmon vibrations in the gold nanoparticles. UV-Vis spectroscopy can be used to record the surface plasmon band (A 540 nm). Husseiny and co-workers also found that an increase in particle size results in a colour shift from pink to blue to yellow. Transmission Electron Microscopy TEM micrographs showing the different shapes of gold nanoparticles produced with the protein expressed in pET28b(+) and by exposing the protein to liquid gold under varying physico-chemical parameters. [Protein] 400 450 500 550 600 650 700 750 800 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 35 (0μg/ml Prot) 37 (10μg/ml Prot) 39 (20μg/ml Prot) 41 (50μg/ml Prot) 43 (30μg/ml Prot) 45 (40μg/ml Prot) Wavelength (nm) Absorbance (A 280 ) [Protein] Blank 400 450 500 550 600 650 700 750 800 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 36 (0μg/mlProt Neg) 38 (10μg/mlProt Neg) 40 (20μg/mlProt Neg) 42 (50μg/mlProt Neg) 44 (30μg/mlProt Neg) 46 (40μg/mlProt Neg) Wavelength (nm) Absorbance (A 280 ) Wave scans on one of the parameters, indicating a change in the ~ 540 nm range due to the difference in particle size and surface plasmon resonance. Mechanism Hypothesis Hypothesis: dithionite reduces the disulfide bonds resulting in a reduced protein which in turn reduces the gold to elemental gold. This was tested by adding dithionite to the assay used as well as iodoacetic acid. Dithionite is a disulfide bond reducing agent and iodoacetic acid is a sulfhydryl blocking agent, therefore preventing the disulfide bonds to become reduced. Thus, if the disulfide bonds in the protein play an integral role in gold reduction, no colour change will be observed. Wave scans and colour changes in the presence and absence of iodoacetic acid. No change in the ~540 nm range occurred and also no change in colour, indicating that the protein wasn’t reduced and therefore no reduction of gold has taken place. Conclusions The ABC transporter, peptide-binding protein were expressed and purified, using the mesophilic host Escherichia coli. Purified protein were able to reduce Au(III) toAu(0), producing nanoparticles with different shapes and sizes, however, formation of these nanoparticles are dependant on environmental parameters and the control of these parameters still needs further investigation. Understanding the mechanism of nanoparticle formations may lead to better candidates for this process and larger protein quantities with the correct folding are needed, therefore expression in a thermophilic host is currently under investigation. Acknowledgements BioPAD, SA; ExBOC, SA; National Research Foundation, SA; University of the Free State, SA; Prof. Derek Litthauer and Dr. Kamini Gounder for the Thermus scotoductus genome database, SA; Prof. Gaetan Borgonie and Myriam Claeys, Ghent University, Belgium. References 1. He, S., Guo, Z., Zhang, Y., Zhang, S., Wang, J. and Gu, N. Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulata. Materials Letters. 61 (2007) 3984-3987. 2. Husseiny, M.I., Abd El-Aziz, M., Badr, Y. and Mahmoud, M.A. Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa. Spectrochimica Acta Part A. 67 (2007) 1003-1006. 3. Mukherjee, P., Senapati, S., Mandal, D., Ahmad, A, Khan, M.I., Kumar, R. and Sastry, M. Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum. ChemBioChem. 5 (2002) 461-463. Dithionite with Iodoacetic acid 400 450 500 550 600 650 700 750 800 0.00 0.05 0.10 0.15 397 (pET28-9) 399 (pET22-7) 398 (pET28-9 Neg) 400 (pET22-7 Neg) Wavelength (nm) Absorbance (A 280 )  pH: 7.4 Buffer: PO 4 - [Protein]: 10 μg/ml [Au]: 2 mM Temp.: 37°C Time: 24 h [Dithionite]: 4.6 μM pH: 7.5 Buffer: PO 4 - [Protein]: 10 μg/ml [Au]: 2 mM Temp.: 65°C Time: 24 h [Dithionite]: 4.6 μM pH: 7.4 Buffer: PO 4 - [Protein]: 10 μg/ml [Au]: 2 mM Temp.: 65°C Time: 24 h [Dithionite]: 4.0 μM pH: 9.5 Buffer: Borax [Protein]: 10 μg/ml [Au]: 2 mM Temp.: 65°C Time: 24 h [Dithionite]: 4.6 μM ABC transporter, peptide binding protein [Reduce Au(III) to Au(0)] pET22b(+) pET28b(+) No His-tag N-terminal His-Tag 0 100 200 300 400 -250 0 250 500 750 mAu mS/cm 40 50 60 ml A 280 (mAu) Conductivity (mS/cm) 0 100 200 300 400 500 -100 0 100 200 300 mAu mS/cm 14 15 16 17 18 ml A 280 (mAu) Conductivity (mS/cm) SDS-PAGE analysis and elution profiles showing the purified protein of about 70 kDa when expressed with the pET22b(+) and pET28b(+) vectors respectively. Dithionite without Iodoacetic acid 400 450 500 550 600 650 700 750 800 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 219 (pET28-9) 380 (pET22-7) 220 (pET28-9 Neg) 382 (pET22-7 Neg) Wavelength (nm) Absorbance (A 280 ) 