Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered sixrhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.Microbial rhodopsins comprise a large family of seven-transmembrane helical proteins that either mediate light-driven ion transport to harvest solar energy or serve as receptors to mediate phototaxis (13) and possibly photoadaptation (32). In archaea, four rhodopsins responding to different wavelengths of light with distinct functions in Halobacterium salinarum have been identified and characterized (20,32 The two ion-transporting rhodopsins perform light-driven outward proton transport to create a proton-electrochemical potential or inward chloride transport to maintain the osmotic and pH homeostasis of the cell. The photoactivated sensory rhodopsins, on the other hand, undergo light-triggered conformational changes to relay signals to their cognate transducers and consequently activate signaling cascades in a manner similar to that of the two-component system involved in eubacterial chemotaxis (1, 22) to control flagellum rotation and thus swimming direction. Our current understanding of microbial rhodopsins as both ion transporters and photosensory receptors has been based primarily on these four known rhodopsins.A recently completed genome project for Haloarcula marismortui (3) proposed the existence of six opsin-related genes, the greatest number ever found in a single archaeon. This proposal immediately raised three questions. (i) Are these six rhodopsins biologically expressed and functionally active? (ii) What is the maximum-absorbance-wavelength ( max ) distribution pattern of these six rhodopsins? (iii) Do the two extra rhodopsins, compared to the four in H. salinarum, perform new functions or have new features beyond those of t...

Practical applicationThis review summarizes recent progress and current status of bioprocess monitoring. There has been an increasing emphasis on the applications of process analytical tools in bioprocessing for biologics manufacturing. The integration of in-line, on-line and at-line sensors and the real-time characterization of the physiological state of cells will lead to robust processes and enhanced product quality. AbstractThe productivity of cell culture manufacturing for biologics has increased momentously in the past decades. Increasingly, the process research efforts are devoted into improving product quality and consistency. Consistent process performance and successful implementation of quality by design (QbD) practice requires well-utilized process analytical technology (PAT). This review summarizes recent progress and current status of bioprocess monitoring. Many sensors for bioprocess monitoring have been available for decades while new ones, especially spectrometric sensors, are making their way into cell culture bioprocesses. On-line sampling devices have grown mature in the past decade thus making many instruments traditionally used for off-line analysis available for at-line use. With a general trend of using better defined medium for cell cultivation and increasing emphasis of process analytical tools, the spectrometric methods are also making headway in cell culture process monitoring. The integration of those sensing technologies will be important to advance the real-time monitoring of the state of cellular physiology for the control for process consistency and product quality.

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system.

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.

Chinese hamster ovary cells, commonly used in the production of therapeutic proteins, are aneuploid. Their chromosomes bear structural abnormality and undergo changes in structure and number during cell proliferation. Some production cell lines are unstable and lose their productivity over time in the manufacturing process and during the product's life cycle. To better understand the link between genomic structural changes and productivity stability, an immunoglobulin G producing cell line was successively single-cell cloned to obtain subclones that retained or lost productivity, and their genomic features were compared. Although each subclone started with a single karyotype, the progeny quickly diversified to a population with a distribution of chromosome numbers that is not distinctive from the parent and among subclones. The comparative genomic hybridization (CGH) analysis showed that the extent of copy variation of gene coding regions among different subclones stayed at levels of a few percent. Genome regions that were prone to loss of copies, including one with a product transgene integration site, were identified in CGH. The loss of the transgene copy was accompanied by loss of transgene transcript level. Sequence analysis of the host cell and parental producing cell showed prominent structural variations within the regions prone to loss of copies. Taken together, we demonstrated the transient nature of clonal homogeneity in cell line development and the retention of a population distribution of chromosome numbers; we further demonstrated that structural variation in the transgene integration region caused cell line instability. Future cell line development may target the transgene into structurally stable regions.