[Methods of preparation of recombinant proteins-cytokines. IV. Renaturation of recombinant human interleukin-3]. / Metody polucheniia recombinantnykh belkov-tsitokinov IV. renaturatsiia rekombinantnogo chelovecheskogo interleikina-3.

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells.

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.

Methods of preparation of recombinant cytokine proteins.

Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.

[Preparation of recombinant cytokines. II. Effective method for isolation, purification and renaturation of human recombinant gamma-interferon]. / Metoddy polucheniya rekombinantnykh belkov-tsitokinov. II*. 'Effektivnyl metod vydeleniya irenaturatsii rekombinantrnogo gamma-interferona cheloveka

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages. A highly active (2 x 10(7) U/mg) recombinant protein of up to 99% purity (according to SDS-PAGE and HPLC) was obtained with a 30% overall yield.