Apparent virus contamination in biopharmaceutical product at centocor.

Out-of-specification (OOS) results were reported by a contract lab in the in vitro adventitious agent assay (AVA) for two products manufactured using mouse myeloma cells in perfusion bioreactors. Cytopathic effect observed for test article-inoculated MRC-5 monolayers resembled foci seen in tissue culture cells infected with transforming viruses. All reasonable known technologies, including highly sensitive, state-of-the-art methodologies and multiple, redundant, and orthogonal methods, were deployed to screen broadly for potential viral and microbial contaminants. Due to the appearance of apparent foci, testing for murine, bovine, and human polyomavirus contamination was heavily represented in the analytical investigation. The results obtained in this extensive screening provided convincing evidence for the lack of an infectious viral or other biological agent. Although the initial investigation produced no reason to invalidate AVA yielding OOS results or to suspect an assay artifact, an extended evaluation revealed several irregularities at the contract test lab reporting the OOS results. The extended investigation also included attempts to reproduce OOS results at alternate contract testing labs and an inter-laboratory study in which methodological differences in the AVA at the three different contract labs were investigated. Only the contract lab initially reporting the OOS results reported foci during this extended evaluation. The results of the inter-laboratory study suggested that the foci artifact might be attributed to the prolonged exposure of the MRC-5 monolayer to cell debris present in the test article. Confocal immunofluorescence microscopy and transmission electron microscopy were subsequently used to provide convincing evidence that the foci observed in test article-inoculated AVA wells were composed of a core of degraded myeloma cell debris covered by one or more layers of MRC-5 cells. The observation that the foci were detected in the AVA at a contract lab where the MRC-5 monolayer is exposed to production cell line debris for a prolonged period strongly suggests that these foci form when MRC-5 grow over the cell debris present in the test article. The cumulative results of the investigation supported the conclusion that the OOS results were artifacts of the AVA test system and not a result of contamination with a virus or other biological agent. Testing was discontinued at the contract lab generating the OOS results and validated at a second contract lab. Manufacturing resumed in consultation with health authorities. The lots were retested following a standard operating procedure (SOP) already in place and ultimately dispositioned for use in normal distribution channels.

Biogenesis of MalF and the MalFGK(2) maltose transport complex in Escherichia coli requires YidC.

The polytopic inner membrane protein MalF is a constituent of the MalFGK(2) maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK(2) maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex.

Contribution of the FtsQ transmembrane segment to localization to the cell division site.

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA.

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the F(1)F(0) ATPase. Recently, it has been shown that the steady state level of the larger and more complex CyoA subunit of the cytochrome o oxidase is also severely affected upon depletion of YidC. In the present study we have analyzed the biogenesis of the integral lipoprotein CyoA. Collectively, our data suggest that the first transmembrane segment of CyoA rather than the signal sequence recruits the signal recognition particle for membrane targeting. Membrane integration and assembly appear to occur in two distinct sequential steps. YidC is sufficient to catalyze insertion of the N-terminal domain consisting of the signal sequence, transmembrane segment 1, and the small periplasmic domain in between. Translocation of the large C-terminal periplasmic domain requires the Sec translocon and SecA, suggesting that for this particular IMP the Sec translocon might operate downstream of YidC.

Reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p involves the nuclear envelope.

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.

Hansenula polymorpha Pex3p is a peripheral component of the peroxisomal membrane.

Hansenula polymorpha Pex3p plays an essential role in the biogenesis and maintenance of the peroxisomal membrane. In the initial report, bakers' yeast Pex3p was suggested to represent an integral component of the peroxisomal membrane, containing one membrane-spanning region that exposes the N terminus of the protein into the organellar matrix. Biochemically, HpPex3p behaved like an integral membrane protein as it was resistant toward high salt and carbonate treatment. However, urea fully removed Pex3p from the membrane under conditions in which the integral membrane protein Pex10p was resistant to this treatment. Additional experiments, including protease protection assays and pre-embedding labeling experiments on purified organellar fractions from cells that produced Pex3ps carrying Myc epitopes at various selected locations in the protein, revealed that invariably all Myc tags were accessible for externally added proteases and antibodies, independent of the presence of detergents. Also, overproduction of Pex3p failed to demonstrate the typical integral membrane protein structures in fracture faces of freeze-fractured peroxisomes. Taken together, our data suggest that HpPex3p does not span the peroxisomal membrane but instead is tightly associated to the cytosolic face of the organelle where it may be present in focal protein clusters.

Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p.

We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p(1-50)) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures. Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles. These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein. We discuss the significance of this finding in relation to peroxisome reintroduction, e.g. in case peroxisomes are lost due to failure in inheritance.

Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration.

The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed.