Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015     

A new regulation of IL-6 production in adult cardiomyocytes by β-adrenergic and IL-1β receptors and induction of cellular hypertrophy by IL-6 trans-signalling

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic β-adrenergic receptor (β-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between β-AR stimulation and pro-inflammatory cytokines, we studied the effects of the β-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G_s protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3^Tyr705 in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that β-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.     

The N terminus of the anti-apoptotic BCL-2 homologue MCL-1 regulates its localization and function

The BCL-2 homologue MCL-1 plays an important role in the regulation of cell fate by blocking apoptosis as well as regulating cell cycle. MCL-1 has an unusual N-terminal extension, which contains a PEST domain and several phosphorylation sites that have been suggested to regulate its turnover. Here we report that the first 79 amino acids of MCL-1 regulate its subcellular localization. Deletion of this domain impairs both its mitochondrial localization and its anti-apoptotic activity. Conversely, expression of the N terminus of MCL-1 promotes both the association of MCL-1 with mitochondria and cell survival in a fashion that is dependent on the presence of endogenous MCL-1. In addition, the N terminus of MCL-1 has an antagonistic effect on proliferation. Although MCL-1 decreases proliferation through binding to proliferating cell nuclear antigen and cyclin-dependent kinase 1 in the nucleus, the N terminus of MCL-1 accelerates cell division. On the other hand, deletion of this region further increases the anti-proliferative activity of MCL-1. These results suggest that the N terminus of MCL-1 plays a major regulatory role, regulating coordinately the mitochondrial (anti-apoptotic) and nuclear (anti-proliferative) functions of MCL-1.     

Skp2B stimulates mammary gland development by inhibiting REA, the repressor of the estrogen receptor

Skp2B, an F-box protein of unknown function, is frequently overexpressed in breast cancer. In order to determine the function of Skp2B and whether it has a role in breast cancer, we performed a two-hybrid screen and established transgenic mice expressing Skp2B in the mammary glands. We found that Skp2B interacts with the repressor of estrogen receptor activity (REA) and that overexpression of Skp2B leads to a reduction in REA levels. In the mammary glands of MMTV-Skp2B mice, REA levels are also low. Our results show that in virgin transgenic females, Skp2B induces lobuloalveolar development and differentiation of the mammary glands normally observed during pregnancy. As this phenotype is identical to what was observed for REA heterozygote mice, our observations suggest that the Skp2B-REA interaction is physiologically relevant. However, in contrast to REA(+/-) mice, MMTV-Skp2B mice develop mammary tumors, suggesting that Skp2B affects additional proteins. These results indicate that the observed expression of Skp2B in breast cancer does contribute to tumorigenesis at least in part by modulating the activity of the estrogen receptor.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Quantifying inbreeding avoidance through extra-pair reproduction

Extra-pair reproduction is widely hypothesized to allow females to avoid inbreeding with related socially paired males. Consequently, numerous field studies have tested the key predictions that extra-pair offspring are less inbred than females’ alternative within-pair offspring, and that the probability of extra-pair reproduction increases with a female's relatedness to her socially paired male. However, such studies rarely measure inbreeding or relatedness sufficiently precisely to detect subtle effects, or consider biases stemming from failure to observe inbred offspring that die during early development. Analyses of multigenerational song sparrow (Melospiza melodia) pedigree data showed that most females had opportunity to increase or decrease the coefficient of inbreeding of their offspring through extra-pair reproduction with neighboring males. In practice, observed extra-pair offspring had lower inbreeding coefficients than females’ within-pair offspring on average, while the probability of extra-pair reproduction increased substantially with the coefficient of kinship between a female and her socially paired male. However, simulations showed that such effects could simply reflect bias stemming from inbreeding depression in early offspring survival. The null hypothesis that extra-pair reproduction is random with respect to kinship therefore cannot be definitively rejected in song sparrows, and existing general evidence that females avoid inbreeding through extra-pair reproduction requires reevaluation given such biases.