Human granulocyte/macrophage colonystimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffnity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radiounmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4-to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coil. The E.coli produced-protein-lacking any carbohydrate-had by far the highest specific activity observed for the recombinant hGM-CSFs.
Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).
Evidence based on analysis of mouse-human somatic-cell hybrids is presented that supports the assignment for the structural loci of peptidase A to human chromosome 18 and of cytoplasmic glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) to chromosome 10. The medical and evolutionary significance of these assignments is discussed.
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