Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

Growth hormone receptor; mechanism of action.

The growth hormone receptor has been an archetype for ligand-induced receptor dimerisation in cytokine receptor signalling. However, we now know that it exists as a constitutive dimer and is activated by a reorganisation of receptor subunits as a result of asymmetric placement of two receptor binding sites on the hormone monomer. This review highlights several topics including: current models of receptor activation; recent advances in the understanding of GH signalling demonstrating that ligand-induced signalling activates Src/ERK pathway in parallel to the classical JAK2-STAT5 signalling; and the nuclear localised growth hormone receptor correlates with high proliferation status and carcinogenesis.

Nuclear targeting of the growth hormone receptor results in dysregulation of cell proliferation and tumorigenesis.

Growth hormone receptor (GHR) has been demonstrated to be nuclear localized both in vivo and in vitro, but the significance of this observation has remained elusive. Here we show that nuclear GHR is strongly correlated with proliferative status in vivo by using a liver regeneration model. In vitro, nuclear translocation of the GH receptor is GH-dependent and appears to be mediated by the Importin system. Constitutive nuclear targeting of GHR in murine pro-B cells is associated with constitutive activation of STAT5, a transforming agent in lymphoma and other cell types. This activation is abrogated by inhibition of JAK2 and appears to be driven by autocrine murine GH action coupled with enhanced nuclear uptake of phospho-STAT5. Nuclear targeting induces dysregulated cell cycle progression in the pro-B cell line, associated with constitutive up-regulation of the proliferation inducers Survivin and Mybbp, the metastasis related Dysadherin, and other tumor markers. GHR nuclear-targeted cells generate aggressive metastatic tumors when injected into nude mice, which display nuclear localized GHR strikingly similar to that seen in human lymphomas. We conclude that aberrant nuclear localization of GHR is a marker of high proliferative status and is sufficient to induce tumorigenesis and tumor progression.

Comparison of three commercial sparse-matrix crystallization screens.

Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.