Production of recombinant proteins by sol-gel immobilized Escherichia coli.

DESIMONE MF, GJ COPELLO, LE DIAZ , C DE MARZI, MM FERNÁNDEZ, EL MALCHIODI, FL PIECKENSTAIN

Badajoz, España

Conferencia; International Conference on Environmental, Industrial and Applied Microbiology; 2005

Formatex Research Center

The mild conditions associated with sol-gel chemistry allow the successful immobilization of a broad range of enzymes but fewer reports involving living cells have been made.  The synthesis of solid inorganic materials from alkoxide and aqueous routes are further areas being developed to improve the viability of encapsulated microorganisms  [1]. 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 three different Escherichia coli BL21 transformants containing plasmid. One contained DNA coding for a T-cell receptor b chain, to be expressed as inclusion body in the cytoplasm, and the other two coding for the bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, for expression as soluble proteins in the periplasm [2].  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 20ºC and the number of viable cells and level of recombinant protein production were analyzed weekly.  Different tests showed that immobilized bacteria conserved their E. coli biochemical characteristics intact.  We found that at both temperatures selected, the number of bacteria in silicon dioxide-derived matrix conserved in the same order of magnitude (109 CFU ml-1) as before immobilization, during 2 months.  Those in an alkoxide-derived matrix decrease to 104 CFU ml-1 at 4ºC and no viable cells were detected at 20ºC after two weeks.  We found that immobilized bacteria could be used as a starter to produce recombinant proteins with a yield comparable with glycerol stocks, 15 mg l-1 for superantigens and 2 mg l-1 for T-cell receptor b chain.  These results contribute to the development of methods for microbial cells preservation under field conditions.