Separation of interleukins by a preparative chromatofocusing-like method.
A chromatofocusing-like method used in the large-scale separation of deamidated from amidated recombinant human interleukin-1 alpha (amino acids 117-271), derived from Escherichia coli, is described. Two major protein species having isoelectric points (pI) of approximately 5.3 and 5.1 were separated by high-performance liquid chromatography using a sulfopropyl strong cation-exchange column. Unlike standard chromatofocusing technique, this method does not use carrier ampholytes during gradient separation of proteins, nor does it employ increased ionic strength for protein elution, the usual method for performing standard ion-exchange chromatography. N-Terminal sequence analysis of the protein with a pI of 5.3 revealed an Asn residue at position 32 as predicted by the cDNA sequence. The pI 5.1 species showed an Asp residue at the same position as a result of deamidation of Asn. This method was also used in the large-scale separation of N-Met from des-Met recombinant human interleukin-1 beta.
Monkarsh
S P
SP
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Russoman
E A
EA
Roy
S K
SK
D002845:Chromatography / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D007375:Interleukin-1 / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D011994:Recombinant Proteins / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D007375:Interleukin-1; D011994:Recombinant Proteins