Effects of Fusion Tags on Protein Partitioning in Aqueous Two-Phase Systems and Use in Primary Protein Recovery

The two techniques aqueous two-phase partitioning and expanded bed adsorption that both are suitable for primary protein recovery were studied. Most of the work was focused on partition in aqueous two-phase systems and in particular on the possibility to effect the partition behaviour by fusion of short peptide tags or protein domains to the target protein. The partitioning of fusion proteins between different variants of the domain tag Z and the naturally occurring protein DNA Klenow polymerase were studied in Breox/Reppal aqueous two-phase systems. Most studies were performed with cell homogenate. The Breox/Reppal system was in focus because if the fusion protein can be partitioned to the Breox-rich top phase the next step can be a thermoseparating aqueous two-phase system. When the Breox phase is heated to 50°C it switches from a one-phase system to a two-phase system resulting in an almost pure water rich top phase and highly concentrated Breox-rich bottom phase. The Breox can then be reused and the protein recovered from the water phase. The Z-domain was genetically modified in different ways to Zbasic1, Zacid2 and Ztrp12 and fused to the Klenow protein to try to enhance partitioning to the Breox-rich phase. From the experiments it was not possible to observe any effects on the partition behaviour irrespectively of tested properties of the domain tag. Despite the absence of domain tag effects high K-values, i.e. partition to the Breox-rich top phase, were observed in the Breox/Reppal system. However, the protein Kvalues seemed to be rather sensitive to the cell homogenate load and showed a tendency to decrease with increased cell homogenate load. Also increased phosphate concentration reduced the K-values. The partitioning of cell debris also seemed to dependent on the cell homogenate load. At higher homogenate load (≤ 20g DW/L) clear Breox-rich top phases were observed with the cell debris collected in Reppal-rich bottom phases. Two different tetrapeptides, AlaTrpTrpPro and AlaIleIlePro were inserted near the Cterminus of the protein ZZT0. The Trp-rich peptide unit strongly increased both the partitioning of ZZT0 into the poly(ethylene glycol) (PEG)-rich phase in a PEG/potassium phosphate aqueous two-phase system and its retention on PEG and propyl hydrophobic interaction chromatographic columns with potassium phosphate as eluent in isocratic systems. Both the partitioning and the retention increased with increasing number of Trp-rich peptide units inserted into ZZT0. Insertion of Ile-rich tetrapeptide units affected the partitioning and retention to a much lesser extent. Partition and modelling data also indicated a folding of inserted Trp and Ile tetrapeptide units, probably to minimise their water contact. It was also investigated how to predict the partitioning of proteins in isoelectric PEG/phosphate aqueous two-phase systems. The capture of ß-galactosidase from E. coli cell homogentate (50g DW/L) by metal chelat expanded bed adsorption was studied. These experiments showed that capture, with a certain degree of selectivity, and clarification of ß-galactosidase could be achieved from a cell homogenate. However, a rather low recovery of about 35 % was obtained at a capacity of 0.25mg/mL of gel. Thus, several parameters remain to be optimised like the load buffer composition and the cell homogenate load.