A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway.

The inability to produce recombinant glycoproteins with authentic N-glycans is a limitation of many heterologous protein expression systems. In the baculovirus-insect cell system, this limitation has been addressed by glycoengineering insect cell lines with mammalian genes encoding protein N-glycosylation functions ("glycogenes") under the transcriptional control of constitutive promoters. However, a potential problem with this approach is that the metabolic load imposed by the expression of multiple transgenes could adversely impact the growth and/or stability of glycoengineered insect cell lines.

Re-visiting the endogenous capacity for recombinant glycoprotein sialylation by baculovirus-infected Tn-4h and DpN1 cells.

It was previously reported that Tn-4h and DpN1 cells have the endogenous capacity to efficiently sialylate secreted alkaline phosphatase (SEAP) when infected with a baculovirus expression vector. In contrast, it has been found that lepidopteran insect cell lines that are more widely used as hosts for baculovirus vectors typically fail to sialylate SEAP and other recombinant glycoproteins. Thus, the N-glycan processing capabilities of Tn-4h and DpN1 cells are of potential interest to investigators using the baculovirus expression system for recombinant glycoprotein production.

Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells.

To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence.

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli.

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient.

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template.

Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus.

Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide.

Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation.

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP.