Avigdor Levanon

Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell

A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.8 so as to solubilize the precipitate and thus the hormone or polypeptide analog contained therein; (e) separating the solubilized hormone or polypeptide analog from other soluble components by gel filtration chromatography; and (f) subjecting the hormone or polypeptide analog thus separated to ion exchange chromatography to purify the hormone or analog and thereby recover purified hormone or polypeptide analog.

Two-Step Sulfate-Enhanced Refolding: Recombinant Pneumocystis carinii Dihydrofolate Reductase*

Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in E. coli where it accumulates in inclusion bodies. The recombinant protein failed to refold after dissolution in high concentrations of denaturants and further dilution in common buffers. A two-step refolding procedure, enabling the enzyme to first assume a partially folded conformation under nonaggregative conditions, and thereafter undergo a further transition to a complete fold, was shown to be indispensable to both regain a reasonable yield of enzymic activity and become a stable protein. Moreover, a simple, inexpensive and easily removable enhancer of refolding, namely Na 2 SO 4 , was found to considerably increase the yield of refolding in the second step. The optimal sulfate concentration was closely dependent on the presence of chloride ions in the refolding buffer. However, to preserve enzymic activity, sulfate had to be removed prior to freezing or lyophilization. Thus, sulfate ions, which have a beneficial effect on refolding, are detrimental to the stability of DHFR, whereas chloride ions have a diametrically opposite effect. Taking advantage of the presence of appropriately located trypto-phanyl residues in P. carinii DHFR, the refolding process could be best monitored by intrinsic fluorescence. The refolded DHFR was also characterized by gel filtration and SDS-PAGE. The final DHFR preparation was >80% pure, could be concentrated up to 2.4 mg/ml, and was stable in solution at 4°C and at—20°C and as a lyophilized powder.