Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

Appl Microbiol Biotechnol (2005) 68: 747–752 DOI 10.1007/s00253-005-1912-7

BIOTECHNOLOG ICA L PROD UCTS A ND PRO CESS ENGINE ERIN G

Martín F. Desimone . Mauricio C. De Marzi . Guillermo J. Copello . Marisa M. Fernández . Emilio L. Malchiodi . Luis E. Diaz

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins Received: 11 November 2004 / Revised: 27 December 2004 / Accepted: 4 January 2005 / Published online: 9 February 2005 # Springer-Verlag 2005

Abstract The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor β chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20°C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (109 cfu ml−1) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 104 cfu ml−1 at 4°C, and no viable cells were detected at 20°C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l−1 for Martín F. Desimone and Mauricio C. De Marzi contributed equally to this work M. F. Desimone . G. J. Copello . L. E. Diaz (*) Cátedra de Química Analítica Instrumental, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 Piso 3°, 1113 Buenos Aires, Argentina e-mail: [email protected] Tel.: +54-11-49648254 Fax: +54-11-49648254 M. C. De Marzi . M. M. Fernández . E. L. Malchiodi Cátedra de Inmunología, IDEHU-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 Piso 4°, 1113 Buenos Aires, Argentina

superantigens and 2 mg l−1 for T-cell receptor β chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

Introduction The increasing use of molecular biology tools and the potential role of bacteria in different biochemical and biotechnological industrial processes make the optimization of microbial cell preservation extremely important. The preservation process must ensure availability of uncontaminated cultures, maintain a high viability rate during the preservation process and storage, and maintain cells in a genetically unmodified state. With knowledge of microbiology techniques, the first objective can be reached easily but the others may present some difficulties. For this reason there are several methods for the preservation of microorganisms but not all of them are suitable under field conditions. One way to increase the duration of preservation could be to immobilize cells in a matrix providing a protective microenvironment that also allows easy recovery and reusage. Sol-gel matrix is a highly porous hydrophilic polymer, providing mechanical strength, and chemical and thermal stability; these characteristics make a sol-gel network suitable for use as a support for immobilization processes. Slight changes in experimental parameters such as pH, additives, and concentration can lead to substantial modification of the resulting supramolecular assembly (Soller-Illia et al. 2002). Different applications of sol-gel technology in environmental biotechnology have been studied (Armon et al. 1996). Sol-gel processes could generate a hydrophilic inorganic matrix that protects immobilized cells against hydrophobic organic solvents (Desimone et al. 2003). Entrapment of biocatalysts in hydrophobic sol-gel materials for use in organic chemistry has been also reported (Reetz 1997). The mild conditions associated with sol-gel chemistry allow the successful immobilization of a broad range of enzymes but there are few reports involving living cells (Branyic et al. 1998; Fennouh et al. 2000; Finnie et al. 2000; Premkumar et al. 2001). The synthesis of solid

748

inorganic materials from alkoxide and aqueous routes are further areas being developed to improve the viability of encapsulated microorganisms (Coiffier et al. 2001). The use of additives such as glycerol and polyethylene glycol, biocompatible pH, and the elimination of cytotoxicity of the silica sol—basically due to alcohol byproducts of alkoxide hydrolysis during the immobilization processes—improves the viability rate after immobilization (Conroy et al. 2000; Nassif et al. 2002). In this way, the synthesis of hybrid silica matrices and the set up of generic tests to assess the viability of Escherichia coli within silica gels have been reported (Armon et al. 2000; Nassif et al. 2003). Development of biocompatible encapsulation for insulin-secreting cells with pores permeable to insulin but not to antibodies, thus avoiding the risk of immune rejection, have also been studied (Pope et al. 1997; Boninsegna et al. 2003). T-cell receptors (TCR) are molecules of the immune system involved in specific antigen recognition on the surface of T-cells. Some viruses and species of bacteria, including Staphylococcus aureus and Streptococcus pyogenes, produce exotoxins called superantigens (SAgs). Bacterial SAgs are 22- to 29-kDa molecules that are heat denaturation and protease resistant and can be absorbed as immunologically intact proteins by epithelial cells (Marrack and Kappler 1990; Hamad et al. 1997). SAgs have the ability to interact with some TCR families in a Vβ restricted manner (Fields et al. 1996; Li et al. 1998), activating a large percentage of the T-cell population (between 5 and 20% of total T-cells), when compared with specific antigen activation (0.05–0.5%). For the purpose of evaluating microbial survival and recombinant protein production in inorganic matrices, we studied the use of silicon oxide matrices for the immobilization and preservation of three E. coli transformants. One was transformed with a chimeric TCR β chain (hVβ5.2mCβ1) expressed as inclusion bodies in the cytoplasm. The other two were transformed with the bacterial SAgs streptococcal superantigen (SSA) and staphylococcal enterotoxin G (SEG), to be expressed as soluble protein in the periplasm. We are proposing an alternative method of long-term preservation without the need for low temperature freezers. This will contribute to microbial cell preservation under field conditions and during transportation, especially in tropical countries. As a model microorganism, we used encapsulated E. coli, the metabolic activity of which remained intact, to produce the specific proteins under study.

Bacterial strains and culture conditions Transformed E. coli BL21 (DE3) from glycerol stocks (hVβ5.2mCβ1, SSA and SEG, cloned in a kanamycinresistant expression vector pET 26b+) were plated on LB agar with 50 μg ml−1 kanamycin and incubated at 37°C overnight. Selected colonies for each transformant were cultured in LB medium with kanamycin up to OD (600 nm) 0.800, centrifuged and resuspended in LB medium with 100 mM sodium phosphate buffer, pH 8.0 and glycerol (20%). The number of colony forming units (cfu) per milliliter of this suspension was determined using the plate count technique prior to its use in immobilization and preservation studies. Immobilization in TEOS The sol was prepared by sonicating (Transsonic 540 sonicator, 35 kHz; Elma, Singen, Germany) a mixture of 1 ml TEOS, 0.2 ml water and 0.06 ml 0.04 M HCl for 30 min at 20°C. After addition of 2 ml water, excess ethanol was eliminated under N2. A bacterial suspension of 2.1×109 cfu ml−1 was mixed with an equal volume of the sol solution. The solution was left for 2 min for gelation. Immobilization in SiO2 The matrix was prepared by acidification to pH 8.0 of a solution of sodium silicate (1.1 g SiO2, 4 ml 2 M NaOH). A bacterial suspension of 2.1×109 cfu ml−1 was mixed with an equal volume of a 5-fold dilution of the sodium silicate solution. Gelation occurred immediately. Determination of viability For determination of viability, decimal dilutions in LB medium of the free E. coli suspension were plated in duplicate in LB agar with and without 50 μg ml−1 kanamycin and incubated at 37°C. After 24 h, the number of cfu was determined. For immobilized cell count, an average weight of 200 mg sol-gel matrix was disrupted using a sterilized glass rod, homogenized in 1 ml LB medium, and used for viable cell determination as for bacterial suspensions. All determinations were made weekly in duplicate over a period of 2 months.

Materials and methods Chemicals

Superantigen production and purification

Tetraethoxysilane (TEOS) and silicon dioxide (SiO2) were from Fluka (Buchs, Switzerland). Luria-Bertani broth (LB) media components and agar from Difco (Kansas City, Mo.) were used for bacterial plate count technique and cultures. All other reagents were of analytical grade.

An LB-agar plate culture with 50 μg ml−1 kanamycin was grown overnight at 37°C from an E. coli BL21 (DE3) glycerol stock. A preculture (20 ml) was grown overnight at 30°C from a single colony or from one sol-gel matrix and then added to 1 l LB culture medium. Bacteria were grown until turbidity (OD 600 nm) reached 0.800 (∼4 h)

749

and were then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) to final concentration of 0.3 mM and cultured for 5 h. The periplasmic fraction, which contained most of the Sag, was obtained by osmotic shock as described previously (Ward 1992; De Marzi et al. 2004). Bacteria were harvested by centrifugation at 7,300 g for 10 min, the pellet resuspended in 50 ml TES buffer (200 mM Tris-HCl pH 8.0, 500 mM sucrose and 0.5 mM EDTA pH 8.0) on ice for 30 min, then centrifuged for 10 min at 12,000 g. The supernatant was kept on ice and the pellet resuspended in 50 ml 5-fold dilution of TES and centrifuged as before. Both supernatants were mixed and dialyzed against PBS. His6-tagged protein was further purified by Ni2+-NTA affinity chromatography as described by the manufacturer (Qiagen, Valencia, Calif.), washed with 20 mM imidazole, 0.5 M NaCl, 0.1 M Tris-HCl pH 8.5 followed by 20 mM imidazole, 0.5 M NaCl, 0.1 M Tris-HCl pH 8.0, then the protein was eluted with 0.3 M imidazole pH 7.5, 10 mM EDTA (Ward 1992). Further purification of SAgs was performed with a size-exclusion Superdex 75 column (Amersham Biosciences, Piscataway, N.J.) equilibrated with 50 mM Tris pH 7.5 150 mM NaCl, and finally purified on a Mono-S cation exchange column (Amersham Biosciences) equilibrated with 50 mM MES pH 6.0 and eluted using a linear NaCl gradient. Protein yield was monitored by measuring absorbance at 280 nm. TCR production and purification An LB-agar plate culture with 50 μg ml−1 kanamycin was grown at 37°C overnight from transformed E. coli BL21 (DE3) glycerol stock. LB medium (1 l) was inoculated with 20 ml overnight culture from glycerol stock or from a solgel matrix, and incubated under agitation at 37°C up to an absorbance of 1.0 at 600 nm. TCR expression was induced with 1 mM IPTG for 4 h. Cells were harvested by centrifugation at 7,000 g for 20 min. The bacterial pellet of hVβ5.2mCβ1 was resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0 and 1 mM DTT) and passed through a French press twice at 1,300 psi (8.96 MPa). The lysate was centrifuged at 7,700 g for 15 min and the inclusion body (IB) pellet was washed four times with Triton X-100 (0.5%) and 100 mM NaCl in lysis buffer. The IB pellet was then washed twice with a solution containing 2 M urea in 2 M NaCl, 50 mM Tris-HCl pH 7.5 and 1 mM DTT, followed by a wash with 4 M urea in the same buffer and, finally, with a solution of 100 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0 and 1 mM DTT. IB were then solubilized in 8 M urea, 100 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0 and 1 mM DTT. The concentration of solubilized IB was estimated in a Coomassie Blue-stained SDS-PAGE gel by comparison with different concentrations of bovine serum albumin (BSA) and then diluted 5-fold in a solution of 6 M guanidine, 10 mM acetate buffer, pH 4.2, and 10 mM EDTA pH 8.0. Denatured β chain was added dropwise to the renaturation buffer (1 M arginineHCl pH 7.5, 2 mM EDTA pH 8.0, 100 mM Tris-HCl pH 7.5,

6.3 mM cysteamine and 3.7 mM cystamina) under vigorous agitation to a final concentration of 30 μg ml−1 at 4°C for 48 h. Refolded hVβ5.2mCβ1 was concentrated and dialyzed extensively against phosphate-buffered saline (PBS) and purified by affinity chromatography using anti-mouse Cβ monoclonal antibody H57-597 (Kubo et al. 1989; Bentley et al. 1995). hVβ5.2mCβ1 was dialyzed against 50 mM MES pH 6.0 and further purified on a Mono-S cationexchange FPLC column (Amersham Biosciences) equilibrated with 50 mM MES pH 6.0 and developed with a linear NaCl gradient. Protein yield was monitored by measuring absorbance at 280 nm. Gel electrophoresis Protein expression was analyzed by SDS-PAGE (12.5%). Whole-cell samples (20 μl) obtained just before induction, and after incubation with IPTG were denatured in SDS buffer and boiled for 5 min prior to electrophoresis. Gel electrophoresis was performed at 140 V in a Bio-Rad Mini Protean cell (Bio-Rad, Hercules, Calif.). The protein gel was then stained for 1 h with Coomassie Blue in 40% methanol and 10% acetic acid. SAgs were also analyzed by immunoblotting using antihistidine monoclonal antibodies (Sigma) or rabbit polyclonal antibodies anti-SSA or anti-SEG, respectively. Rabbit polyclonal antisera were obtained by immunization with 10 μg SSA or SEG mixed with complete Freud’s adjuvant. The boost was administered on days 7, 14 and 28. Sera obtained on day 35 were tested by 10-fold serial dilutions in ELISA and immunoblotting procedures. Biochemical characterization The biochemical properties of free and sol-gel immobilized E. coli BL21 (DE3) were determined by standard methods such as Levin Broth, Cromogenic Agar CPS, glucose fermentation, glucose gas, citrate, indole, Methyl Red, Voges Proskauer, LIA, motility and β glucuronidase. Biochemical characterization was carried out following standard methods typically applied in clinical microbiology and were performed in order to detect any alteration in the characteristic biochemical pathways of the immobilized bacteria. These biochemical reactions are not supposed to be quantitative techniques but were used simply to characterize bacterial phenotypes.

Results Immobilized E. coli BL21 (DE3) was stored in sealed tubes at 4°C and 20°C for 2 months, during which the number of viable cells and level of recombinant protein production were analyzed. Viability studies showed that E. coli cells survived the immobilization process in the sol-gel network.

750 Table 2 Bacterial preservation at 4 and 20°C for silicon dioxide (SiO2)- and tetraethoxysilane (TEOS)-derived matrices. The number of cfu for SSA producers E. coli BL21 (DE3) was determined in LB agar with 50 μg ml−1 kanamycin at 37°C for 24 h Matrix

TEOS SiO2

Fig. 1 Viability of an encapsulated Escherichia coli streptococcal superantigen (SSA) transformant maintained at 4°C after immobilization in tetraethoxysilane (TEOS; ▲)- and silicon dioxide (SiO2; ■)-derived matrices as determined in LB plates with kanamycin

As shown in Fig. 1, 72 h after the encapsulation process, the amount of live bacteria was similar to that in the original suspension in both matrices employed. These determinations were made in LB plates with and without kanamycin and no significant differences were found in cfu number during the 60 days assayed, indicating that the bacteria conserve the antibiotic resistance expression vector. All immobilized E. coli BL21 (DE3) transformants also preserved the biochemical characteristics of wild type E. coli, being positive for Levin Broth, cromogenic agar CPS, glucose fermentation, glucose gas, indole, Methyl Red, LIA, motility and β glucuronidase, and negative for citrate and Voges Proskauer. As can be seen in Fig. 1, viability of immobilized SSAproducing bacteria in SiO2-derived matrices remained mostly constant at 4°C until the end of the experiment 60 days later. In contrast, bacteria immobilized in TEOSderived matrix, showed a decrease in survival, until no viable cells were found on day 42. As shown in Table 1, similar results were obtained with SEG and TCR β chain transformants when immobilized in SiO2. The TEOS matrix, however, was not able to preserve bacteria beyond day 35. In addition, we studied the viability of the im-

cfu/ml at 4°C

cfu/ml at 20°C

Day 3

Day 60

Day 3

Day 60

1.5×109 2.1×109

0 1.7×108

2.1×108 1.3×109

0 5.1×108

mobilized clones at 20°C, finding a death kinetic higher than at 4°C for TEOS-derived matrix but, in SiO2 encapsulated bacteria, viability remains almost on the same level as the initial suspension, as shown in Table 2 for SSA producers. Similar results were found for SEG and TCR β chain producers (not shown). These determinations were also made in LB plates with and without kanamycin and no significant differences were found during the 60 days assayed. Bacteria immobilized in SiO2 matrix were used weekly as a starter for 1 l LB medium culture, and recombinant protein production was analyzed by 12.5% SDS-PAGE. As can be seen in Fig. 2, the pattern of regular protein expression from E. coli is similar in non-encapsulated and in encapsulated cells for 60-day bacteria. In addition, Fig. 2 shows the presence of a characteristic band in SDS-PAGE corresponding to either SEG or the TCR β chain (both are compared with purified SSA since all these proteins have a similar molecular weight of ∼28 kDa). Both transformants showed high expression of recombinant protein in TEOSand SiO2-derived matrices despite possessing different expression systems that direct protein to two very different environments: IB in the cytoplasm or soluble protein in the periplasm. Expression of recombinant protein produced by SiO2encapsulated E. coli was assessed by treatment of bacteria encapsulated for 60 days with sample buffer for SDS-PAGE and subsequent immunoblotting. As shown in Fig. 3, antihistidine tag monoclonal antibody and specific anti-SEG

Table 1 Viability of different Escherichia coli BL21 (DE3) transformants encapsulated in SiO2 and maintained at 4°C. The number of colony forming units (cfu) was determined in LB agar with and without 50 μg ml−1 kanamycin at 37°C for 24 h. SSA Streptococcal superantigen, SEG staphylococcal enterotoxin G, TCR T-cell receptor Protein producer clone

SSA SEG TCR β chain

cfu/ml (day 3)

cfu/ml (day 60)

LB LB +kanamycin

LB LB +kanamycin

2.1×109 1.5×108 2.1×108

1.9×109 1.6×108 1.8×108

1.7×108 2.3×107 4.1×107

1.8×108 2.2×107 3.8×107

Fig. 2 Protein expression by encapsulated bacteria. Bacteria were used as a starter for a 1 l LB medium culture and the expression of recombinant protein was analyzed by 12.5% SDS-PAGE. Wholecell lysates from bacteria encapsulated for 60 days were used for the analysis. Lanes: a Purified superantigen (SAg) SSA (shown as molecular weight marker); b, e samples taken before induction with IPTG; c, f samples taken after induction of encapsulated bacteria; d, g samples taken after induction of non-encapsulated bacteria. TCR T-cell receptor β chain, SEG staphylococcal enterotoxin G

751

Fig. 3a,b Immunoblot analysis of SAg SEG expression. Whole-cell lysates from bacteria encapsulated for 60 days were used for the analysis. Lanes: a Encapsulated bacteria, b non-encapsulated bacteria, c non-induced bacteria, d FPLC-purified SEG. Anti-histidine tag monoclonal antibody (a) and anti-SEG rabbit polyclonal antibodies (b) were used as primary antibody

rabbit polyclonal antibody were able to recognize the specific recombinant protein in the complex mixture of induced bacteria, whereas bands were absent in non-induced controls. The protein yields for SAgs and TCR β chain were ∼15 mg l−1 and ∼2 mg l−1, respectively, which are very similar to yields obtained in our laboratory from cultures starting from glycerol stocks.

Discussion Since immobilization or adsorption on inert matrices may cause alterations in the metabolic behavior of cells, as described in several studies revealing altered metabolic regulation, increased ethanol tolerance (Desimone et al. 2002), and modifications in membrane fatty acid composition (Hilge-Rotmann and Rehm 1991), more information is necessary on the physiology of immobilized cells, especially for those that produce specific products in which genetic stability and intact metabolic pathways are relevant. The results presented here show that immobilization in a matrix obtained from SiO2 precursors is effective in the

preservation of transformed E. coli with high viability rates and without contamination. This SiO2-derived particulate matrix with immobilized bacteria generates a suitable microenvironment that can be efficiently stored at either 4 or 20°C in sealed tubes without contamination for at least 60 days. Moreover, the use of glycerol, which works as an osmotic stabilizer and also prevents excessive contraction of sol-gel matrices, protects cells against immobilization stress. The viability rate was similar in both matrices 72 h after immobilization, but the TEOS-derived matrix was unable to preserve bacterial survival for longer than that. Moreover, the protocol followed for immobilization in TEOS-derived matrix, in which a bacterial suspension in sodium phosphate buffer is added in the second step of the reaction, avoids exposure to acid and ethanol. It was also hypothesized that the presence of residual ethanol from further hydrolysis and condensation reactions could possibly explain the lower viability detected in alkoxide-derived polymeric matrices during the period assayed. Similarly, the use of nitrogen to eliminate ethanol produced by hydrolysis of TEOS prior to the addition of the bacterial suspension, generates an anaerobic medium that could contribute to bacterial susceptibility. Anaerobicity contributes to bacterial susceptibility only during the immobilization process; thereafter, the porous matrix allows biochemical exchange with the oxygen present at atmospheric pressure. Although wet TEOS-derived matrices had better mechanical properties, it was difficult to completely eliminate the ethanol generated during the polymerization reaction; this drawback was absent in SiO2-derived matrices. The amount of water in the wet silica gels was constant during the period assayed since they were stored in sealed tubes. At the onset of the experiment, the nutrient concentration was the same for both matrices, but bacterial access may differ due to the properties of the matrices—particulate for SiO2-derived and polymeric for TEOS-derived matrices (Brinker and Scherer 1990). Residual ethanol and nutrient accessibility may be involved in the differential viability found. The use of the plate count technique and the evaluation of specific protein production after immobilization of the cells provides a suitable correlation between the number of viable cells and their production capability. Although a proportion of the bacteria would be too tightly trapped to be plated, leading to a lower counting result, the use of more drastic methods may cause lethal damage, thus affecting cfu determination. If an alternate assay technique, such as measuring enzyme activity, was used, the results could remain constant or even become higher due to cell lysis after cell immobilization. The metabolic activity of the cells must remain intact in order to produce the specific proteins under study. Protein yields from bacteria immobilized in SiO2-derived matrices were the same as that obtained from glycerol stocks for the three molecules under study. In addition, the recombinant proteins were recognized by specific antibodies, confirming the preservation of the interaction sites and, thus, preservation of protein structure.

752

We conclude that bacteria immobilized in SiO2-derived matrices remain genetically stable, preserve their antibiotic resistance, and recombinant protein production capability. This work contributes to the study of new matrices with the capacity to conserve different bacterial strains for use in biochemical or biotechnological processes. Immobilization of microorganisms for different applications not only has the advantage of conserving their viability, but also facilitates storing conditions without external contamination. Acknowledgements We thank R. Langley for critical reading of the manuscript. M.F.D. is grateful for his doctoral fellowship granted by Universidad de Buenos Aires. M.C.D. is grateful for his doctoral fellowship granted by CONICET. E.L.M. and L.E.D. are supported by grants from Universidad de Buenos Aires; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Investigaciones Científicas y Técnicas. L.E.D. and E.L.M. are members of CONICET research career.

References Armon R, Dosoretz C, Starosvetsky J, Orshansky F, Saadi I (1996) Sol-gel applications in environmental biotechnology. J Biotechnol 51:279–285 Armon R, Starosvetzky J, Saad I (2000) Sol-gel as a reaction matrix for bacterial enzymatic activity. J Sol-Gel Sci Technol 19:289– 292 Bentley GA, Boulot G, Karjalainen K, Mariuzza RA (1995) Crystal structure of the beta chain of a T-cell antigen receptor. Science 267:1984–1987 Boninsegna S, Bosetti P, Carturan G, Dellagiacoma G, Dal Monte R, Rossi M (2003) Encapsulation of individual pancreatic islets by sol–gel SiO2: a novel procedure for perspective cellular grafts. J Biotechnol 100:277–286 Branyic T, Kuncova G, Paca J, Demmerova K (1998) Encapsulation of microbial cells into silica gel. J Sol-Gel Sci Technol 13:283– 287 Brinker CJ, Scherer GW (1990) Sol-Gel Science. The physics and chemistry of sol-gel processing. Academic, San Diego Coiffier A, Coradin T, Roux C, Bouvet O, Livage J (2001) Sol-gel encapsulation of bacteria: a comparison between alkoxide and aqueous routes. J Mater Chem 11 2039–2044 Conroy JFT, Power ME, Martin J, Earp B, Hosticka B, Daitch CE, Norris PM (2000) Cells in sol-gels I: a cytocompatible route for the production of macroporous silica gels. J Sol-Gel Sci Technol 18:268–283

De Marzi MC, Fernández MM, Sundberg EJ, Molinero L, Zwirner NW, Llera AS, Mariuzza RA, Malchiodi EL (2004) Cloning, expresión and interaction of human T-cell receptors with bacterial superantigen SSA. Eur J Biochem 271:4075–4083 Desimone MF, Degrossi J, D’Aquino M, Diaz LE (2002) Ethanol tolerance in free and sol-gel immobilised Saccharomyces cerevisiae. Biotechnol Lett 24:1557–1559 Desimone MF, Degrossi J, D’Aquino M, Diaz LE (2003) Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media. Biotechnol Lett 25:671–674 Fennouh S, Guyon S, Livage J, Roux C (2000) Sol-gel entrapment of Escherichia coli. J Sol-Gel Sci Technol 19:647–649 Fields BA, Malchiodi EL, Li H, Isern X, Stauffacher CV, Schlievert PM, Karjalainen K, Mariuzza RA (1996) Crystal structure of a T cell receptor β chain complexed with a superantigen. Nature 384:188–192 Finnie KS, Bartlett JR, Woolfrey JL (2000) Encapsulation of sulfate reducing bacteria in a silica host. J Mater Chem 10:1099–1101 Hamad AR, Marrack P, Kappler JW (1997) Transcytosis of staphylococcal superantigen toxins. J Exp Med 185:1447–1454 Hilge-Rotmann B, Rehm HJ (1991) Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae. Appl Microbiol Biotechnol 34:502–508 Kubo RT, Born JW, Kappler P, Marrack P, Pigeon M (1989) Characterization of a monoclonal antibody which detects all murine αβ T cell receptor. J Immunol 142:2736–2742 Li H, Llera A, Tsuchiya D, Leder L, Ysern X, Schlievert PA, Karjalainen K, Mariuzza R (1998) Three-dimensional structure of the complex between a T cell receptor β chain and the superantigen Staphylococcal enterotoxin B. Immunity 9:807– 816 Marrack P, Kappler J (1990) The staphylococcal enterotoxins and their relatives. Science 248:705–711 Nassif N, Bouvet O, Rager MN, Roux C, Coradin T, Livage J (2002) Living bacteria in silica gels. Nat Mater 1:42–44 Nassif N, Roux C, Coradin T, Rager MN, Bouvet OMM, Livage J (2003) A sol-gel matrix to preserve the viability of encapsulated bacteria. J Mater Chem 13:1–7 Pope EJA, Braun K, Peterson CM (1997) Bioartificial organs I: silica gel encapsulated pancreatic islets for the treatment of diabetes mellitus. J Sol-Gel Sci Technol 8:635–639 Premkumar JR, Lev O, Rosen R, Belkin S (2001) Encapsulation of luminous recombinant E. coli in sol-gel silicate films. Adv Mater 13:1773–1775 Reetz MT (1997) Entrapment of biocatalysts in hydrophobic sol-gel materials for use in organic chemistry. Adv Mater 9:943–954 Soller-Illia GJAA, Sanchez C, Lebeau B, Patarin J (2002) Chemical strategies to design textured materials: from microporous and mesoporous oxides to nanonetworks and hierarchical structures. Chem Rev 102:4093–4138 Ward ES (1992) Secretion of T-cell receptor fragments from recombinant Escherichia coli cells. J Mol Biol 224:885–890