Maria Ehn

Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

A Multi-disciplinary Approach in the Development of a Stroke Rehabilitation Tool

This work describes a method used in the development of a stroke rehabilitation tool. The method was based on three key elements. The first key element was iterations between the use of broad groups with different professionals/stakeholders and small hands-on working groups with users from the same profession. The second key element was movement between understanding differences between different organizations and professionals and understanding of specific needs within the different organizations. The final key element was including implementation aspects from the very start of the work.

Activity Monitors as Support for Older Persons’ Physical Activity in Daily Life: Qualitative Study of the Users’ Experiences

Background Falls are a major threat to the health and independence of seniors. Regular physical activity (PA) can prevent 40% of all fall injuries. The challenge is to motivate and support seniors to be physically active. Persuasive systems can constitute valuable support for persons aiming at establishing and maintaining healthy habits. However, these systems need to support effective behavior change techniques (BCTs) for increasing older adults’ PA and meet the senior users’ requirements and preferences. Therefore, involving users as codesigners of new systems can be fruitful. Prestudies of the user’s experience with similar solutions can facilitate future user-centered design of novel persuasive systems. Objective The aim of this study was to investigate how seniors experience using activity monitors (AMs) as support for PA in daily life. The addressed research questions are as follows: (1) What are the overall experiences of senior persons, of different age and balance function, in using wearable AMs in daily life?; (2) Which aspects did the users perceive relevant to make the measurements as meaningful and useful in the long-term perspective?; and (3) What needs and requirements did the users perceive as more relevant for the activity monitors to be useful in a long-term perspective? Methods This qualitative interview study included 8 community-dwelling older adults (median age: 83 years). The participants’ experiences in using two commercial AMs together with tablet-based apps for 9 days were investigated. Activity diaries during the usage and interviews after the usage were exploited to gather user experience. Comments in diaries were summarized, and interviews were analyzed by inductive content analysis. Results The users (n=8) perceived that, by using the AMs, their awareness of own PA had increased. However, the AMs’ impact on the users’ motivation for PA and activity behavior varied between participants. The diaries showed that self-estimated physical effort varied between participants and varied for each individual over time. Additionally, participants reported different types of accomplished activities; talking walks was most frequently reported. To be meaningful, measurements need to provide the user with a reliable receipt of whether his or her current activity behavior is sufficient for reaching an activity goal. Moreover, praise when reaching a goal was described as motivating feedback. To be useful, the devices must be easy to handle. In this study, the users perceived wearables as easy to handle, whereas tablets were perceived difficult to maneuver. Users reported in the diaries that the devices had been functional 78% (58/74) of the total test days. Conclusions Activity monitors can be valuable for supporting seniors’ PA. However, the potential of the solutions for a broader group of seniors can significantly be increased. Areas of improvement include reliability, usability, and content supporting effective BCTs with respect to increasing older adults’ PA.

Users perspectives on interactive distance technology enabling home-based motor training for stroke patients.

The aim of this work has been to develop a technical support enabling home-based motor training after stroke. The basis for the work plan has been to develop an interactive technical solution supporting three different groups of stroke patients: (1) patients with stroke discharged from hospital with support from neuro team; (2) patients with stroke whose support from neuro team will be phased out and (3) patients living with impaired motor functions long-term. The technology has been developed in close collaboration with end-users using a method earlier evaluated and described [12]. This paper describes the main functions of the developed technology. Further, results from early user-tests with end-users, performed to identify needs for improvements to be carried out during further technical development. The developed technology will be tested further in a pilot study of the safety and, usefulness of the technology when applied as a support for motor training in three different phases of the post-stroke rehabilitation process.