Ribosomes and subunits from Escherichia coli studied by asymmetrical flow field-flow fractionation
Sammanfattning: In this thesis asymmetrical flow field-flow fractionation (FFF) separations of ribosomes and subunits have been studied. Ribosomal material was prepared from Escherichia coli (E. coli) cells and analyzed by the size separation technique. The five main peaks in the fractograms correspond to transfer RNA (tRNA), unidentified proteins, small subunit, large subunit, and ribosome. The accuracy of the method was tested by measuring the mass fraction of ribosome and the number of ribosomes per cell. Both parameters changed in an expected manner and changes larger than 5 and 10%, respectively, are significant. Furthermore, the amount of tRNA per cell was determined. The method has been used for elucidating the effect of Vitreoscilla hemoglobin (VHb) on the translational capacity of the host-cell, E. coli. The expression of hemoglobin increased both the number of ribosomes per cell and the amount of tRNA per cell during oxygen-limited fed-batch cultivation. However, the mass fraction of ribosome was unaffected by the expression of VHb. The increase in the number of ribosomes was accompanied by an increase in the activity of a recombinant marker protein, beta-lactamase. The use of the separation method in the optimization of protein production by genetically engineered E. coli cells was evaluated. A correlation between protein yield and the number of ribosomes per cell was found. The mass fraction of ribosomes was also correlated to the protein yield. These correlations could be used for eliminating the need of determining the protein yield in optimization studies. Instead, the method development can be performed using asymmetrical flow FFF to determine the ribosomal parameters.
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