The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate‐binding subunits or domains in order to couple the energy‐providing reaction with transport. In the maltose transporter of several Gram‐negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate‐binding subunit contains a C‐terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 Å resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2‐fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

We report the crystallization and structure determination at 1.85 A Ê of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-af®nity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modi®cation. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel b-sheet¯anked by a-helices on both sides. Both are connected by a hinge region consisting of two antiparallel b-strands and an a-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Speci®cally, we ®nd helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

BackgroundSingle-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.ResultsCultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.ConclusionsRocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

During the past five years, the number of single‐use bioreactors used in biopharmaceutical research and production has increased tremendously. This increase has been particularly associated with mammalian cell culture processes from small‐ to medium‐scale volumes. Even though nowadays customers can choose from a multitude of 2nd and 3rd generation single‐use bioreactors, ranging from mL‐ up to m3‐scale, there is a lack of knowledge of their engineering parameters. Different approaches have been applied to characterization investigations, resulting in an inability to compare different single‐use bioreactors with each other and their reusable counterparts, creating an obstacle to a systematic approach to scaling‐up the process. This article describes parametric, experimental and computer‐based numeric methods for biochemical engineering characterization of single‐use bioreactors, which have already been used successfully for the characterization of their reusable counterparts. For the first time, these methods have been evaluated in terms of their practical application.

In 2004, the FDA published a guideline to implement process analytical technologies (PAT) in biopharmaceutical processes for process monitoring to gain process understanding and for the control of important process parameters. Viable cell concentration (VCC) is one of the most important key performance indicator (KPI) during mammalian cell cultivation processes. Commonly, this is measured offline. In this work, we demonstrated the comparability and scalability of linear regression models derived from online capacitance measurements. The linear regressions were used to predict the VCC and other familiar offline biomass indicators, like the viable cell volume (VCV) and the wet cell weight (WCW), in two different industrially relevant CHO cell culture processes (Process A and Process B). Therefore, different single-use bioreactor scales (50–2000 L) were used to prove feasibility and scalability of the in-line sensor integration. Coefficient of determinations of 0.79 for Process A and 0.99 for Process B for the WCW were achieved. The VCV was described with high coefficients of determination of 0.96 (Process A) and 0.98 (Process B), respectively. In agreement with other work from the literature, the VCC was only described within the exponential growth phase, but resulting in excellent coefficients of determination of 0.99 (Process A) and 0.96 (Process B), respectively. Monitoring these KPIs online using linear regression models appeared to be scale-independent, enabled deeper process understanding (e.g. here demonstrated in monitoring, the feeding profile) and showed the potential of this method for process control.