2-STEP CHROMATOGRAPHIC PROCEDURE FOR PURIFICATION OF BASIC FIBROBLASTGROWTH-FACTOR FROM RECOMBINANT ESCHERICHIA-COLI AND CHARACTERIZATION OF THE EQUILIBRIUM PARAMETERS OF ADSORPTION

A two-step chromatographic procedure for purification of basic fibrobl ast growth factor (bFGF) from high-cell-density cultures of recombinan t E. coli is described. Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for pu rification of bFC-F from the soluble cell fraction. A one-step affinit y chromatographic procedure resulted in very pure bFGF. However; this one-step affinity isolation of bFGF caused the loss of around 60% of t he recombinant protein. A combination of ion-exchange chromatography w ith heparin-Sepharose affinity chromatography was favored for bFGF pur ification. A first cation-exchange chromatographic step resulted in a solution of bFCF with a purity of around 70%. The weak cation exchange r CM-Sepharose C50 was preferred in comparison to the strong cation ex changer S-Sepharose because of the higher recovery of bFGF. With the i on-exchange chromatographic step prior to the heparin-Sepharose affini ty chromatography, the total yield of recovery of bFGF increased to 56 % compared to 40% using the one-step purification procedure with hepar in-Sepharose. To characterize the equilibrium parameters of adsorption , batch experiments for the calculation of maximum capacities and diss ociation constants for CM-Sepharose C50 and heparin-Sepharose were car ried out. The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction accordin g to the Langmuir model of adsorption. The adsorption of bFGF to hepar in-Sepharose was described by a double Langmuir approach of two indepe ndent binding sites with different maximum capacities and dissociation constants. The purified bFGF showed a high biological activity and ci rcular dichroic spectra of a proper folded molecule. The analysis of t he N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first ami no acid methionine. In addition, half of the bFGF molecules lacked the second amino acid alanine.