Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli.

This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies. These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations. They exhibit full in vitro biological activity and have slightly lower pI's. Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively. The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis. These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.

Screening for a "new" enzyme in nature: Haloperoxidase production by Death Valley dematiaceous hyphomycetes.

Haloperoxidases are enzymes that have the ability to halogenate a broad range of substrates [10]. To find a biologically produced haloperoxidase that could function at a pH greater than 3.0 and at a temperature greater than 19°C, dematiaceous hyphomycetes were isolated from the Death Valley desert and screened for their ability to produce such an enzyme. A qualitative assay using bromophenol red was employed in situ over a 12-day fermentation period. Several dematiaceous hyphomycetes, such asDreschlera haloides andUlocladium chartarum, produced haloperoxidases that were active in broth culture at 19, 25, and 34°C at pH 7.0 and 8.0.