Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-γ but not of soluble human interleukin-2 by the Tat pathway in Escherichia coli

"Production of periplasmic human interferon-γ (hINF-γ) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-γ was processed and the protein was transported to periplasm. Up to 30.1% of hINF-γ was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.

Optimization of human interferon gamma production in Escherichia coli by response surface methodology

"The production of human interferon gamma (hIFN-γ) using a synthetic gene in Escherichia coli BL21-SI was optimized by response surface methodology (RSM) and a Box-Behnken design. The process variables studied were temperature, bio-mass concentration at induction time and the NaCl concentration as inducer. According to the Box-Behnken design, a second order response function was developed. The optimal expression conditions were a temperature of 32.6°C, induction biomass of 0.31 g/L and 0.3 M NaCl in minimal medium. The model prediction for the maximum hIFN-γ production was 77.3 mg/L, which corresponded satisfactorily with the experimental data. The hIFN-γ concentration attained under optimized conditions was 13-times higher than that obtained using the non-optimized conditions. We conclude that RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology: the case of human interferon beta

"A human interferon beta (hINF-β) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-β production. The maximum hINF-β production of 61 mg l−1 was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 °C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes.

System for expressing and transporting recombinant proteins to the escherichia coli periplasm using the Tat (twin-arginine translocation) secretion pathway

"La presente invención describe un vector de expresión que contiene un péptido señal que utiliza la vía de secreción Tat como alternativa al sistema Sec de secreción para el transporte de citocininas y otras proteínas de interés biotecnológico al periplasma de Escherichia coli. La parte novedosa de esta invención consiste en utilizar el péptido señal de la penicilino acilasa (SPpac) mutado y fusionado al gen sintético de interferón-? humano (inf.-?) para el transporte de la proteína al periplasma de Escherichia coli por vía Tat. El SPpac mutado contiene el sitio NdeI en el extremo 5', conteniendo en el vector de expresión pEMR, y en el extremo 3' el codón de triptófano se cambia por el codón de serina y con ello se genera el sitio de restricción Hind III. Se modifican los codones de Leucenia y Alanina para generar el sitio NheI. Con ello se generan dos sitios de restricción para la clonación y fusión en fase de genes de proteínas homólogas ó heterólogas. Actualmente, no hay vectores comerciales disponibles para la expresión y transporte de proteínas mediante péptidos señal de vía de transporte Tat.""The present invention describes an expression vector which contains a signal peptide using the Tat secretion pathway as an alternative of the Sec secretion system for the transport of cytoquinine and further proteins of biotechnological interest to Escherichia coli periplasm. The novel system consists in using the signal peptide of penicillin acylase (SPpac) which is mutated and fussed to the synthetic human interferon-y gene (hINF-y) for the transport of the protein to the Escherichia coli periplasm using the Tat pathway. The mutated SPpac includes the NdeI site at the 5 end, which is contained in the expression vector pEMR, at the 3 end the tryptophan codon being exchanged by the serine codon, thus generating the restriction site Hind III. The Leucine and Alanine codons are modified so as to generate the NheI site. Thus, two sites for restricting the cloning and fusion within a gene phase of homologous or heterologous proteins are generated. Nowadays there is no commercial vector available for the expression and transport of proteins, which include signal peptides using the Tat pathway.