| 1. |
- Berggren, Kristina, et al.
(författare)
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Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems
- 1999
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 62:2, s. 135-144
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135-144, 1999.
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| 2. |
- Carlsson, Mats, et al.
(författare)
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Effects of fused tryptophan rich peptides to a recombinant protein A domain on the partitioning in polyethylene glycol-dextran and Ucon-dextran aqueous two-phase systems
- 1996
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 756:1-2, s. 107-117
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used to construct fusion proteins with tryptophan containing peptides. The peptides and the fusion proteins have been partitioned in aqueous two-phase systems of poly(ethylene glycol) (PEG)-dextran and Ucon-dextran. The studied model protein was ZZT0, where Z is an engineered domain of domain B of staphylococcal protein A. The specially designed hydrophobic peptides, Ala-Trp-Trp-Pro (T1) and (Ala-Trp-Trp-Pro)2 (T2), have been inserted into ZZT0, to give the peptide-protein fusions ZZT1 and ZZT2. In the experimental studies it was found that T1 and T2 preferred the PEG phase and even more the Ucon phase over the dextran phase. For T2 the partitioning was more one sided than for T1. For the fusion proteins, ZZT1 and ZZT2, the partitioning was enhanced into the PEG or Ucon rich phase as compared to ZZT0. The effects were lower than expected from independent contributions to the partition coefficient from the protein and the peptides. A heterogeneous lattice model was used to calculate theoretical peptide and protein partition coefficients. The calculations could reproduce the qualitative features of the experimental data. The model results suggest that a part of these experimentally observed effects is due to a depletion zone, i.e. a zone of reduced polymer concentration around the protein. The experimental results indicate a further reduction of the partition coefficient, beyond that predicted by the lattice calculations. A possible folding of the inserted peptide is discussed as a plausible mechanism for this further reduction in the partition coefficient.
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| 3. |
- Svensson, Mårten, et al.
(författare)
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Aqueous two-phase systems containing self-associating block copolymers - Partitioning of hydrophilic and hydrophobic biomolecules
- 1999
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 839:1-2, s. 71-83
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Tidskriftsartikel (refereegranskat)abstract
- A series of proteins and one membrane-bound peptide have been partitioned in aqueous two-phase systems consisting of micelle-forming block copolymers from the family of Pluronic block copolymers as one polymer component and dextran T500 as the other component. The Pluronic molecule is a triblock copolymer of the type PEO-PPO-PEO, where PEO and PPO are poly(ethylene oxide) and poly(propylene oxide), respectively. Two different Pluronic copolymers were used, P105 and F68, and the phase diagrams were determined at 30oC for these polymer systems. Since the temperature is an important parameter in Pluronic systems (the block copolymers form micellar-like aggregates at higher temperatures) the partitioning experiments were performed at 5 and 30oC, to explore the effect of temperature-triggered micellization on the partitioning behaviour. The temperatures correspond to the unimeric (single Pluronic chain) and the micellar states of the P105 polymer at the concentrations used. The degree of micellization in the F68 system was lower than that in the P105 system, as revealed by the phase behaviour. A membrane-bound peptide, gramicidin D, and five different proteins were partitioned in the above systems. The proteins were lysozyme, bovine serum albumin, cytochrome c, bacteriorhodopsin and the engineered B domain of staphylococcal protein A, named Z. The Z domain was modified with tryptophan-rich peptide chains in the C-terminal end. It was found that effects of salt dominated over the temperature effect for the water-soluble proteins lysozyme, bovine serum albumin and cytochrome c. A strong temperature effect was observed in the partitioning of the integral membrane protein bacteriorhodopsin, where partitioning towards the more hydrophobic Pluronic phase was higher at 30oC than at 5oC. The membrane-bound peptide gramicidin D partitioned exclusively to the Pluronic phase at both temperatures. The following trends were observed in the partitioning of the Z protein. (i) At the higher temperature, insertion of tryptophan-rich peptides increased the partitioning to the Pluronic phase. (ii) At the lower temperature, lower values of K were observed for ZT2 than for ZT1.
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