Biomedical subjects

T Q Faria

Publications and source records attributed to T Q Faria.

3 recordsLinked to original sources

PubMed2006

Relationship between retroviral vector membrane and vector stability.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Animals↗

PubMed2001

Cutinase unfolding and stabilization by trehalose and mannosylglycerate.

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.

Carboxylic Ester Hydrolases↗

PubMed2000

High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli.

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.

Calorimetry, Differential Scanning↗