Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells.

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-¹³C-glucose and ¹5N-glutamate as labeled precursors. This study suggests that uniformly ¹5N,¹³C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

All-or-none N-glycosylation in primate follicle-stimulating hormone beta-subunits.

Human FSH exists as two major glycoforms designated, tetra-glycosylated and di-glycosylated hFSH. The former possesses both alpha- and beta-subunit carbohydrates while the latter possesses only alpha-subunit carbohydrate. Western blotting differentiated the glycosylated, 24,000 M(r) hFSHbeta band from the non-glycosylated 21,000 M(r) FSHbeta band. Postmenopausal urinary hFSH preparations possessed 75-95% 24,000 M(r) hFSHbeta, while pituitary hFSH immunopurified from 21- to 43-year-old females and 21-43-year-old males possessed only 35-40% 24,000 M(r) hFSHbeta. The pituitary hFSH from a postmenopausal woman on estrogen replacement was 75% 21,000 M(r) hFSHbeta. Other immunopurified postmenopausal pituitary hFSH preparations possessed 50-60% 21,000 M(r) hFSHbeta. Gel filtration removed predominantly 21,000 M(r) free hFSHbeta and reduced its abundance to 13-22% in postmenopausal pituitary hFSH heterodimer preparations. A major regulatory mechanism for FSH glycosylation involves control of beta-subunit N-glycosylation, possibly by inhibition of oligosaccharyl transferase. Two primate species exhibited the same all-or-none pattern of pituitary FSHbeta glycosylation.

An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells.

It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with 15N and carbohydrates with 13C by NMR spectroscopy.

Bartonella henselae in porpoise blood.

We report detection of Bartonella henselae DNA in blood samples from 2 harbor porpoises (Phocoena phocoena). By using real-time polymerase chain reaction, we directly amplified Bartonella species DNA from blood of a harbor porpoise stranded along the northern North Carolina coast and from a pre-enrichment blood culture from a second harbor porpoise. The second porpoise was captured out of habitat (in a low-salinity canal along the northern North Carolina coast) and relocated back into the ocean. Subsequently, DNA was amplified by conventional polymerase chain reaction for DNA sequencing. The 16S-23S intergenic transcribed spacer region obtained from each porpoise was 99.8% similar to that of B. henselae strain San Antonio 2 (SA2), whereas both heme-binding phage-associated pap31 gene sequences were 100% homologous to that of B. henselae SA2. Currently, the geographic distribution, mode of transmission, reservoir potential, and pathogenicity of bloodborne Bartonella species in porpoises have not been determined.

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells.

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.