Tesis doctoral de Carvajal Vallejos Patricia Karina
In order to overexpress maize transglutaminase in a heterologous system, the first cloned plant transglutaminase from maize (tgz) cdnas were subcloned into the e. Coli expression vector pet28. Four constructs (tgz4, tgz15, tgz9 and tgz29) with different sequence characteristics were obtained. the overexpression of the recombinant tgz4p in e. Coli bl21 de3 cells cultured under standard conditions produced active protein in the soluble fraction, however the majority of protein was found in inclusion bodies. The overexpressed tgz proteins were identified as zea mays tg by maldi-tof analysis. With respect to the different constructs, those lacking the chloroplast transit peptide signal yielded more tgzp. two synthetic culture media (m9+glucose and m9+glycerine) with the same carbon quantity as the reference medium lb, were designated. The µ values and biomass concentrations obtained in minimal media were lower than lb medium in erlenmeyer flasks. Monod equation parameters for e. Coli bl21 de3 harbouring the tgz4 gene cultured in m9+ glucose were evaluated. bioreactor experiments in batch mode (5l) showed that iptg induction and temperature downshift did no stop cellular growth, however decreased the cellular growth rate. After the analysis of recombinant protein production along the culture time, the maximum tgz4p accumulation was detected after 2 to 3 hours of iptg induction. bath and fplc denatured purification, obtained recombinant protein of higher purity and quantity than native purification. Tgz4p and e. Coli co-purified proteins were identified by maldi-tof analysis. The peptide mass fingerprint (pmf) analysis covered 71% of the tgz4p sequence. After selection of the adequate tgz4p refolding buffer, functional tgzp was obtained. Purified tgz4p was also used to obtain specific tgz4p polyclonal antibodies which quantitatively give more signal than the t7·tag and pep antibodies for transmission electron microscopy tgz4p immunolocal
Datos académicos de la tesis doctoral «Zea mays l. transglutaminase expression in escherichia coli«
- Título de la tesis: Zea mays l. transglutaminase expression in escherichia coli
- Autor: Carvajal Vallejos Patricia Karina
- Universidad: Ramón llull
- Fecha de lectura de la tesis: 02/06/2006
Dirección y tribunal
- Director de la tesis
- Eduard Barber? Moral
- Tribunal
- Presidente del tribunal: miquel Gassiot matas
- José María Torne cubiro (vocal)
- Antonio Planas sauter (vocal)
- pablo Fuentes- prior (vocal)