C. Mark Smales

Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems

Although the cold-shock response has now been studied in a number of different organisms for several decades, it is only in the last few years that we have begun to understand the molecular mechanisms that govern adaptation to cold stress. Notably, all organisms from prokaryotes to plants and higher eukaryotes respond to cold shock in a comparatively similar manner. The general response of cells to cold stress is the elite and rapid overexpression of a small group of proteins, the so-called CSPs (cold-shock proteins). The most well characterized CSP is CspA, the major CSP expressed in Escherichia coli upon temperature downshift. More recently, a number of reports have shown that exposing yeast or mammalian cells to sub-physiological temperatures (<30 or <37 degrees C respectively) invokes a co-ordinated cellular response involving modulation of transcription, translation, metabolism, the cell cycle and the cell cytoskeleton. In the present review, we summarize the regulation and role of cold-shock genes and proteins in the adaptive response upon decreased temperature with particular reference to yeast and in vitro cultured mammalian cells. Finally, we present an integrated model for the co-ordinated responses required to maintain the viability and integrity of mammalian cells upon mild hypothermic cold shock.

Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D‐PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post‐protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D‐PAGE can be used for monitoring and identification of HCPs post‐protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell‐engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251.

Biochemical insights into the mechanisms central to the response of mammalian cells to cold stress and subsequent rewarming

Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27–32 °C) but became increasingly disrupted at low temperatures (4–10 °C). The degradation rates of all mRNAs and proteins examined were much reduced at 27 °C, and overall protein synthesis rates were gradually reduced with temperature down to 20 °C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27 °C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4 °C), lipid‐containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery.

Host cell protein adsorption characteristics during protein A chromatography.

Protein A chromatography is a critical and ‘gold‐standard’ step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI‐TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back‐bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D‐PAGE was then used to determine individual components associated with resin back‐bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification.

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mm glucose and at 37 °C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a α-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the α-helix, forcing the region of the helix around the site of glycation to adopt a 310 helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.

Evaluation of individual protein errors in silver-stained two-dimensional gels

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error.

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells.

Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co‐expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over‐expression of the yeast ‘protein disaggregase’ Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over‐expression of the yeast or mammalian Hsp70/40 chaperones. Over‐expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5‐fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over‐expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over‐expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells. Biotechnol. Bioeng. 2010; 105: 556–566.

Post-Translational Events of a Model Reporter Protein Proceed With Higher Fidelity and Accuracy Upon Mild Hypothermic Culturing of Chinese Hamster Ovary Cells

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37 degrees C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32 degrees C but was diminished at lower temperatures. The expression of both cap-dependent and cap-independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32 degrees C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post-translational events appear to proceed with higher fidelity and accuracy at 32 than 37 degrees C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32 degrees C recombinant cap-dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post-translational processing.

Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates.

Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein‐based drugs. The establishment of high‐producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post‐translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed. Biotechnol. Bioeng. 2009;102: 1593–1602.