Improvement in the heterogeneous N‐termini and the defective N‐glycosylation of human interleukin‐6 by genetic engineering

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Abstract
Recombinant human interleukin‐6 (IL‐6), expressed in Chinese hamster ovary cells, has heterogeneous N‐termini of Ala1 and Va13, as does naturally occurring IL‐6. This heterogeneity is thought to be caused by difficulty in cleavage of the signal sequence. To obtain homogeneous IL‐6, Pro at ‐1 was exchanged for A1a by site‐directed mutagenesis. Alternatively, the signal sequence was replaced with that of human granulocyte‐colony‐stimulating factor. In both cases, the IL‐6 designed to start with Ala1 was still heterogeneous, while the IL‐6 designed to start with Val3 showed a homogeneous N‐terminus. It is suggested that the heterogeneity of the N‐terminus is caused not only by the signal sequence, but also by the succeeding sequences of the mature protein. Only a portion of recombinant human IL‐6 is N‐glycosylated. Asn46, being exchanged for Gln by site‐directed mutagenesis, was confirmed to be partially N‐glycosylated. The defective N‐glycosylation was assumed to be caused by interference or tension from a disulfide bond near the N‐glycosylation site. To verify this hypothesis, the Cys45 and Cys51 forming the disulfide bond were exchanged for Ser. The N‐glycosylated species became predominant upon this substitution, suggesting that formation of the disulfide bond is a cause of the defective N‐glycosylation.