Principal Investigator (PI): Prof Sophia Hober

Personnel: Tove Alm (PhD-student), Karin Larsson (PhD-student, shared with H Wernérus), Johanna Steen (PhD-student, shared with J Ottosson), Hanna Tegel (PhD-student, shared with J Ottosson); Cecilia Eriksson (PhD-student), Anna Konrad (PhD-student), Johan Nilvebrant (PhD-student)

Fig 1. Green Fluorescent Protein (GFP) is a versatile reporter protein, which can be used for assessment of protein solubility in vivo, as a basis for monitoring the production of recombinant proteins under different conditions.

Summary and objectives: The focus of the group is to develop predictable and robust systems for protein production, purification and detection.
Despite extensive work and attempts to relate protein folding to amino acid no reliable method exists for prediction of propensity to misfold in vivo. Therefore we have developed a reliable high throughput method to screen protein expression of both soluble and precipitated protein using a flow cytometer. Through gene fusion of target proteins to a solubility reporter protein (eGFP), the whole cell fluorescence and forward scattered light can be used to assess the protein production in terms of relative levels of soluble product and inclusion body formation. The method is used for optimization of protein production. Moreover we are working with well-characterized, small and folded domains descending from the bacterial receptors staphylococcal protein A (SPA) or streptococcal protein G (SPG). Known three-dimensional structures, a capability of independent folding and the absence of disulfide bridges make these domains ideal as frameworks for further protein engineering. In the current research we are using protein-engineering strategies in order to custom make proteins for protein purification processes. The goal with the mutagenesis could be either to stabilize or destabilize ligands for different purposes. We are also working with designed domains with extreme surface charge. These proteins allow for ion exchange chromatography under conditions favorable for selective and efficient capture of fused target proteins. These domains have been used for purification in native and denaturating conditions as well as solid phase refolding. Combinatorial strategies are also employed to randomize certain areas of the proteins in order to construct domains capable of selective recognition of new targets. The designed purification tags and antibodies produced within the HPR program are combined to develop strategies for capture of proteins and protein complexes in an efficient and selective.


Fig 2. Model of the electrostatic potentials of three engineered variants of the B domain from protein A. The domain is engineered to accommodate different amounts of negative charge.