Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation.

Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.

The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize (Zea mays).

Prolamellar bodies (PLB) contain two photochemically active forms of the enzyme protochlorophyllide oxidoreductase POR-PChlide640 and POR-PChlide650 (the spectral forms of POR-Chlide complexes with absorption maxima at the indicated wavelengths). Resuspension of maize PLB in media with a pH below 6.8 leads to a rapid conversion of POR-PChlide650 to POR-PChlide640 and a dramatic re-organization of the PLB membrane system. In the absence of excess NADPH, the absorption maximum of the POR complex undergoes a further shift to about 635 nm. This latter shift is reversible on the re-addition of NADPH with a half-saturation value of about 0.25 mm NADPH for POR-PChlide640 reformation. The disappearance of POR-PChlide650 and the reorganization of the PLB, however, are irreversible. Restoration of low-pH treated PLB to pH 7.5 leads to a further breakdown down of the PLB membrane and no reformation of POR-PChlide650. Related spectral changes are seen in PLB aged at room temperature at pH 7.5 in NADPH-free assay medium. The reformation of POR-PChlide650 in this system is readily reversible on re-addition of NADPH with a half-saturation value about 1.0 microm. Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide650, membrane organization and NADPH binding. The low-pH driven spectral changes seen in maize PLB are shown to be accelerated by adenosine AMP, ADP and ATP. The significance of this is discussed in terms of current suggestions of the possible involvement of phosphorylation (or adenylation) in changes in the aggregational state of the POR complex.