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We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Mammalian cells are often used for the expression of recombinant proteins. The process of screening transfected cells randomly for high producing clones is tedious and time consuming. We evaluated using green fluorescent protein (GFP) for selection of high producing clones by fluorescence-activated cell sorter (FACS) to reduce screening effort. We expressed neurotrophin-3 (NT3), deoxyribonuclease (DNase), or vascular endothelial growth factor (VEGF) with GFP in Chinese hamster ovary cells. The vector expressed the desired secreted protein and the selectable marker, dihydrofolate reductase, in one expression unit and the intracellular GFP in a second expression unit. Transfected cells were grown in selection medium and sorted by FACS. High fluorescence clones were obtained and found to produce high amounts of the desired protein; VEGF productivity correlated well with GFP fluorescence in 48 clones. Further studies demonstrated that productivity correlated very well with RNA of the desired protein. For comparison, we randomly picked and screened 144 VEGF clones, and the highest producing VEGF clone obtained produced 0.7 pg/cell/day. In contrast, the highest producing VEGF clone obtained by FACS sorting produced 4.4 pg/cell/day. FACS sorting therefore selected high producing clones efficiently. Since an assay for the desired protein is not required, high producing clones for a protein of unknown function can be obtained by FACS sorting followed by measuring the RNA level of the desired protein in the highly fluorescent clones.

We have constructed expression vectors for Chinese hamster ovary (CHO) cells that produce both selectable marker and recombinant cDNA from a single primary transcript via differential splicing. These vectors produce stable CHO cell clones that, when pooled, produce abundant amounts of secreted recombinant proteins compared with the amounts produced by conventional expression approaches that have selectable marker and the cDNA of interest under control of separate transcription units. Our vectors divert most of the transcript to product expression while linking it, at a fixed ratio, to dihydrofolate reductase (DHFR) expression to allow selection of stable transfectants. Pools of clones with increased expression of the product gene can be efficiently generated by selection in methotrexate. The high level of expression from pools allows convenient and rapid production of milligram amounts of recombinant proteins.

We have cloned the gene coding for a ubiquitin-protein hydrolase from the yeast Saccharomyces cerevisiae. The gene (YUH1) was isolated from a yeast genomic library by screening with an oligonucleotide probe designed from the amino acid sequence of the purified hydrolase. The YUH1 gene encodes a 26 kD protein and contains no introns. The YUH1 gene product can be overexpressed in active form in Escherichia coli and purified by a two column procedure. The purified hydrolase is capable of cleaving ubiquitin-protein fusions in vitro specifically at the ubiquitin fusion junction and requires no high energy cofactors. Fusions can also be cleaved in E. coli in strains expressing the hydrolase. Gene disruptions in haploid yeast strains have no apparent phenotypic change and ubiquitin-protein hydrolase activity in extracts is normal, indicating the existence of additional genes for ubiquitin-protein hydrolase. In vitro and in vivo cleavage of ubiquitin-protein fusions may be a useful method of producing proteins with defined ammo-termini.