| 1. |
- Binz, Hans, et al.
(författare)
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Method for enhancing the immunogenicity of an immunogenic compound or hapten, and use thereof for preparing vaccines
- 1994
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Patent (populärvet., debatt m.m.)abstract
- The present invention relates to a process for improving the immunogenicity of an immunogen, an antigen or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said antigen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to mammalian serum albumin. The invention also relates to the use, as a medicament, of the product which can be obtained in this way.
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| 2. |
- Binz, Hans, et al.
(författare)
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Respiratory syncytial virus protein g expressed on bacterial membrane
- 1994
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Patent (populärvet., debatt m.m.)abstract
- A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.
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| 3. |
- Eklund, M., et al.
(författare)
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Anti-idiotypic protein domains selected from protein A-based affibody libraries
- 2002
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 48:3, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fe binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K.) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.
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| 4. |
- Gräslund, Torbjörn, et al.
(författare)
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Charge engineering of a protein domain to allow efficient ion-exchange recovery
- 2000
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 13:10, s. 703-709
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Tidskriftsartikel (refereegranskat)abstract
- We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.
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| 5. |
- Gräslund, Torbjörn, et al.
(författare)
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Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
- 2002
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 96:1, s. 93-102
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Tidskriftsartikel (refereegranskat)abstract
- The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.
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| 6. |
- Gräslund, Torbjörn, et al.
(författare)
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Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
- 1997
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 9, s. 125-132
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Tidskriftsartikel (refereegranskat)abstract
- A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.
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| 7. |
- Gräslund, Torbjörn, et al.
(författare)
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Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 942:1-2, s. 157-166
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Tidskriftsartikel (refereegranskat)abstract
- 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 α-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≈5.8) and ZZ-Cutinase-Zbasic (pI≈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.
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| 8. |
- Gulich, S., et al.
(författare)
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Stability towards alkaline conditions can be engineered into a protein ligand
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 169-178
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Tidskriftsartikel (refereegranskat)abstract
- One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.
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| 9. |
- Ljungqvist, Charlotta, et al.
(författare)
-
Derivatives of the B or Z domain from staphylococcal protein A (SPA) interacting with at least one domain of human factor VIII
- 1999
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Patent (populärvet., debatt m.m.)abstract
- The present invention relates to modified polypeptides which are derivatives of the B domain or Z domain from staphylococcal protein A (SPA), wherein between 1 and 20 amino acid residues of the said B or Z domain have been substituted by other amino acid residues, said substitution being made without substantial loss of the basic structure and stability of the said B or Z domain, and said substitution resulting in interaction capacity of the said polypeptide with at least one domain of human Factor VIII protein.
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| 10. |
- Ljungqvist, Charlotta, et al.
(författare)
-
Receptor structures
- 1999
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Patent (populärvet., debatt m.m.)abstract
- The present invention relates to modified polypeptide derivatives of the B domain or Z domain of staphylococcal protein A (SPA). The derivatives contain amino acid substitutions that result in the ability of the B domain or Z domain to interact with at least one domain of a human Factor VIII protein, without substantially disrupting the structure and stability of the B domain or Z domain.
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