High-level expression and production of recombinant human interleukin-6 analogs.
We have constructed and analyzed different mutant forms of interleukin-6 (IL-6) expressed in Escherichia coli that can be divided into two groups. The first group contains four full-length IL-6 molecules that differ in the presence of cysteine residues involved in disulfide bridges. The second group contains 22 N-terminal amino acid deletions in addition to the differences in the cysteine residues. The different IL-6 muteins were extracted and their expression levels and solubility were compared. We found that the production levels of IL-6 can be dramatically improved by deleting the first 22 N-terminal amino acids of the molecule. We have also found that the production of IL-6 containing the four cysteine residues is lower than the production of the mutant molecules that lack one or both pairs of cysteines. The yield of soluble and properly refolded IL-6 was the highest when the disulfide bond between the cysteines at positions 74 and 84 was present in the mutein form, which also lacked the 22 N-terminal amino acids.
Dagan
S
S
Department of Molecular Biology, ImClone Systems Incorporated, New York, New York 10014.
Tackney
C
C
Skelly
S M
SM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D015870:Gene Expression; D015850:Interleukin-6 / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D015850:Interleukin-6; D011994:Recombinant Proteins; D003545:Cysteine