Scalable transient protein expression.

Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. This review will summarize developments in this field, properties of the components of a transient expression system that enable maximal protein production, and detailed protocols for this application.

Proliferation and pluripotency of human embryonic stem cells maintained on type I collagen.

Human embryonic stem cells (hESC) require a balance of growth factors and signaling molecules to proliferate and retain pluripotency. Conditioned medium (CM) from a human embryonic germ-cell-derived cell culture, SDEC, was observed to support the growth of hESC on type I collagen (COL I) and on Matrigel (MAT) biomatricies. After 1 month, the population doubling of hESC grown in SDEC CM on COL I was equivalent to that of hESC grown in mouse embryonic fibroblast (MEF) CM on MAT. hESC grown in SDEC CM on COL I expressed OCT4, NANOG, SSEA-4, alkaline phosphatase (AP), and TRA-1-60; retained a normal karyotype; and were capable of forming teratomas. DNA microarray analysis was used to compare the transcriptional profiles of SDEC and the less supportive WI38 and Detroit 551 human cell lines. The mRNA level of secreted frizzled-related protein (sFRP-1), a known antagonist of the WNT/ß-catenin signaling pathway, was significantly reduced in SDEC as compared with the other 2 cell lines, whereas the mRNA levels of prostaglandin-endoperoxide synthase 2 (PTGS2 or COX-2) and prostaglandin I2 synthase (PGIS), two prostaglandin biosynthesis genes, were significantly increased in SDEC. The level of sFRP-1 protein was significantly reduced, and levels of 2 prostaglandins that are downstream products of PTGS2 and PGIS, prostaglandin E2 and 6-keto-prostaglandin F(1α), were significantly elevated in SDEC CM compared with WI38, Detroit 551, and MEF CM. Further, addition of purified sFRP-1 to SDEC CM reduced the proliferation of hESC grown on COL I as well as MAT in a dose-dependent manner.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells.

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man(5)GlcNAc(2)-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man(9)GlcNAc(2)-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man(5)GlcNAc(2)-PP-Dol through Glc(1)Man(9)GlcNAc(2)-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc(3)Man(9)GlcNAc(2)-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life.

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.