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.

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6 percent and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1 percent and a purification factor of 3.90 for the second, and a recovery of 76.2 percent and a purification factor of 2.36 for the last one. The overall yield was 50 percent, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84 percent pure as analyzed by HPLC.

Purification of bovine pancreatic glucagon as a by-product of insulin production.

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC.

Competitive inhibition by the D-isomer in racemic mixtures used as substrate in kinetic studies: a simple method for data treatment.

A set of equations was applied that allows the use of racemic mixtures for the estimation of kinetic parameters in systems where the L-isomer is substrate and the D-isomer a competitive inhibitor, displaying data as double reciprocal plots. A statistical treatment was introduced that renders compatible the output of presently available programs with the specific model requirements, with few additional calculations. An example is shown with beta-trypsin and its inhibition of benzozyl-D-arginine p-nitroanilide (Ki = 1.12 mM, pH 8,0, 37 degrees C).

Alpha- and beta- rat urinary kallikreins: chemical and physicochemical properties.

Alpha- and beta- urinary kallikreins were isolated by affinity chromatography on Sepharose-L-Arg-OMe (E. Silva, C.R. Diniz and M. Mares-Guia, Biochemistry, 13: 4304-4310, 1974). Both enzymes lowered rat arterial blood pressure and contracted the rat uterus. Alpha- and beta-kallikreins consist of single polypeptide chains of 27900 and 24900 daltons and have sedimentation coefficients of 2.49 S and 2.55 S, respectively. The alpha enzyme has 18 amino acid residues more than the beta enzyme and their carbohydrate content is 11.9% and 7.75%, respectively. Neuraminidase treatment removed all sialic acid residues from both forms and this improved their stability in aqueous solution at pH 7.0, 37 degrees C. After electrophoresis on 12% polyacrylamide gels at pH 8.3, followed by activity measurements of gel slices with H-D-Val-Leu-Arg-pNA, two active bands each were detected for alpha and beta-kallikrein, but in different proportions and with different specific activity. On the basis of the rat uterus assay, alpha-kallikrein was three times more active than beta-kallikrein. The amino acid composition of alpha-kallikrein is strikingly similar to that of pig kallikrein B and human urinary kallikrein.