Descrição
The ability to cultivate microbial strains, expressing heterologous recombinant
proteins, is an increasingly important technique in biotechnology. Among the
many systems available Escherichia coli remains one of the most attractive due
to its ability to grow rapidly and at high densities on inexpensive substrates, its
well-characterized genetics and the availability of an increasingly large number of
cloning vectors and mutant host strains. However, the recombinant proteins
produced with E. coli are usually very difficult to measure and so the sampling
points along a fermentation run are not sufficient to elucidate some bottlenecks
that could exist and are not suitable for process control, modeling and
optimization purposes. In this work, a factorial experimental design was used to
optimise three variable culture conditions of a model system based on the
expression of EYFP protein (Enhanced Yellow Fluorescent Protein): operational
temperature, dissolved oxygen and of induction time. During the E. coli
fermentation, samples were taken every hour for evaluation of standard state
variables. Furthermore, the protein concentration was analysed by
spectroflorimetry with no sample processing. A western blotting technique was
used to validate the fluorimetric measurements and to determine the subcellular
localization of EYFP protein. Other tests were also performed for the validation of
this methodology, as linearity analysis and effect of the presence of biomass.
; Rocha, I. 
