PubMed · 10503526
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T Shimizu. 1999. [Acetate].. https://pubmed.ncbi.nlm.nih.gov/10503526/
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Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli. In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus. VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E. coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E. coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR.
Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubule-stimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.
A number of recent studies have presented perspectives on the hydrophobic fluorescence probe tryptophan octyl ester (TOE). This molecule has attracted notable attention as a suitable model for the natural fluorophore tryptophan, in case of membrane proteins. We report here, for the first time, the fluorescence emission behaviour of TOE in reverse micelles of aerosol-OT (AOT) in n-heptane, containing different amounts of water. Relevant studies in representative homogeneous solvent media are also included for comparison. The fluorescence emission parameters (especially emission maximum, relative intensity, and anisotropy) of TOE are found to exhibit significant variation upon changes in the water/surfactant molar ratio (w(0)) of the reverse micelles. Fluorescence decay studies on TOE which we have also performed, indicate biexponential decay kinetics in reverse micelles as well as in homogeneous solvent media. The implications of these findings are examined in relation to the potentialities of TOE as a novel fluorescence probe for membrane proteins present in water restricted environments prevailing at the interfaces of biomembranes (for which reverse micelles serve as ideal model systems).