5 research outputs found
A structurally informed autotransporter platform for efficient heterologous protein secretion and display.
<p>Abstract</p> <p>Background</p> <p>The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the <it>Escherichia coli</it> AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the <it>Mycobacterium tuberculosis</it> vaccine target ESAT6 as a model protein.</p> <p>Results</p> <p>Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of <it>E. coli</it>. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated <it>Salmonella typhimurium</it> strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.</p> <p>Conclusions</p> <p>We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.</p
Spatial and nonspatial implicit motor learning in Korsakoff’s amnesia: evidence for selective deficits
Patients with amnesia have deficits in declarative memory but intact memory for motor and perceptual skills, which suggests that explicit memory and implicit memory are distinct. However, the evidence that implicit motor learning is intact in amnesic patients is contradictory. This study investigated implicit sequence learning in amnesic patients with Korsakoff’s syndrome (N = 20) and matched controls (N = 14), using the classical Serial Reaction Time Task and a newly developed Pattern Learning Task in which the planning and execution of the responses are more spatially demanding. Results showed that implicit motor learning occurred in both groups of participants; however, on the Pattern Learning Task, the percentage of errors did not increase in the Korsakoff group in the random test phase, which is indicative of less implicit learning. Thus, our findings show that the performance of patients with Korsakoff’s syndrome is compromised on an implicit learning task with a strong spatial response component
Scientific discovery learning with computer simulations of conceptual domains
Scientific discovery learning is a highly self-directed and constructivistic form of learning. A computer simulation is a type of computer-based environment that is well suited for discovery learning, the main task of the learner being to infer, through experimentation, characteristics of the model underlying the simulation. In this article we give a review of the observed effectiveness and efficiency of discovev learning in simulation environments together with problems that learners may encounter in discovery learning, and we discuss how simulations may be combined with instructional support in order to overcome these problems