| Autor: |
R V, Tikhonov, S E, Pechenov, I A, Belacheu, S A, Yakimov, V E, Klyushnichenko, E F, Boldireva, V G, Korobko, H, Tunes, J E, Thiemann, L, Vilela, A N, Wulfson |
| Rok vydání: |
2001 |
| Předmět: |
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| Zdroj: |
Protein expression and purification. 21(1) |
| ISSN: |
1046-5928 |
| Popis: |
Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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