Stabilized, long-term expression of heterodimeric proteins from tricistronic mRNA.

A major problem in the use of recombinant mammalian cells for protein overexpression is their long-term stability, in particular, when the foreign gene product exerts a negative effect on the producer cells. We have addressed this issue and developed a vector system for the stable expression of heterodimeric recombinant proteins in mammalian cells. In this system, the two recombinant cDNAs and the puromycin-resistant gene are transcribed as a single tricistronic transcript. An efficient translation of the internal cistrons is mediated by internal ribosome entry sites between them. On the example of expression of a heterodimeric antibody fusion protein in BHK-21 cells, we show that the translational coupling of the antibody genes to the selectable marker in a tricistronic expression construct allows long-term stabilization of expression by continuous application of selection pressure. This vector system allows fast and straightforward construction of expression plasmids for the generation of producer cell lines, even for complex heterodimeric proteins with unlimited long-term stability.

Anatomy of highly expressing chromosomal sites targeted by retroviral vectors.

The eukaryotic genome contains chromosomal loci with a high transcription-promoting potential. For their identification in cultured cells, transfer of a reporter gene has to be performed by a technique that grants the integration of individual copies. We have applied retroviral vectors in conjunction with inverse polymerase chain reaction techniques to reconstruct a number of these sites for a further characterization. Remarkably, all examples conform to the same design in that the process of retroviral infection selected a scaffold- or matrix-attached region (S/MAR) that was flanked by DNA with high bending potential. The S/MARs are of an unusual type in that they show a high incidence of certain dinucleotide repeats and the potential to act as topological sinks. The anatomy of retroviral integration sites reveals principles that can be exploited for the development of predictable transgenic systems on the basis of expression and targeting vectors.

Regulation of cell growth by IRF-1 in BHK-21 cells.

Most cell lines that are used for the production of recombinant proteins proliferate spontaneously at a high rate. In many types of cultivation systems these cells still keep growing after having reached the desired cell density. Further proliferation in batch cultures leads to cell death as a consequence of nutrient and oxygen depletion as well as to accumulation of lactate and toxic products. Consequently, in many technical processes, the surplus of cells is removed.We have established cell lines in which proliferation is controlled by a physiological regulator, IRF-1. IRF-1 (Interferon Regulatory Factor 1) is a transcriptional activator and acts as a tumor suppressor. Constitutive overexpression of recombinant IRF-1 leads to inhibition of cell growth. The extent of this growth arrest depends on the intracellular concentration of active IRF-1. To allow IRF-1 expression in various mammalian cells a system for conditional IRF-1 activation has been established. A fusion protein composed of IRF-1 and the hormone binding domain of the human estrogen receptor, was used. This system allows to control gradually the growth of several mammalian cell lines by adjusting the intracellular concentration of active IRF-1 via estradiol in the medium. We have evaluated BHK-21 cells with respect to IRF-1 mediated cell growth inhibition and expression of two secreted proteins. Whereas the productivity of proliferation inhibited cells with respect to constitutively transcribed IgG genes is reduced, productivity of another secreted protein which is controlled by an IRF-1 inducible promoter is strongly enhanced under these conditions.

Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources.

The genetic stability of human cell lines in long-term culture has been tested by DNA fingerprinting a panel of 31 different continuous cell lines from patients with leukemias or lymphomas. Duplicates of the same cell line obtained from different sources, subclones of cell lines, and samples of cell lines at different passage levels were studied. In most cases the fingerprints of duplicates of the same cell line remained perfectly preserved even after long-time passaging. However, in five cases there were notable differences between individual fragments of corresponding fingerprints. We have found four cases of mislabeled and/or cross-contaminated cell lines so far. Taken together, our results indicate that DNA fingerprinting qualifies as a very reliable means of cell line identification which allows the detection of mislabelling or contamination and of genetic variation among subclones.

Sensitivity and specificity of five different mycoplasma detection assays.

The sensitivity and specificity of five different mycoplasma detection tests were evaluated in comparison with the classical microbiological culture assay on agar plates as the reference method: direct fluorochrome DNA staining (direct DAPI), DNA staining of an indicator cell line (indirect DAPI), RNA hybridization with a cDNA specific for ribosomal mycoplasmal RNA, an enzyme-linked immunosorbent assay (ELISA) with mycoplasma-specific antibodies, and a biochemical cytotoxicity assay (6-MPDR). A large panel of continuous cell lines (20 adherent and 233 suspension cell lines, most of the latter were human leukemia-lymphoma cell lines) were analyzed for infection with mycoplasma. The results of the comparative analysis for sensitivity and specificity of the various tests were as follows: 100% and 100% for the indirect DAPI, 100% and 98% for the RNA hybridization assay, 87% and 94% for the direct DAPI, 72% and 100% for the ELISA, 75% and 90% for the biochemical 6-MPDR assay. Each of these approaches has both advantages and disadvantages with regard to cost, time, reliability, specificity, and sensitivity. The best compromise for routine mycoplasma testing is a combination of several techniques (e.g. direct culture on agar, RNA hybridization, and direct or indirect DAPI).

An exported protein of Plasmodium falciparum is synthesized as an integral membrane protein.

Exp-1 is an antigen of Plasmodium falciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranous compartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-1 in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its N-terminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp-1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm.

In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum.

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.

Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers.

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and single-stranded template is discussed.