N-glycosylated human growth hormone with prolonged circulatory half-life

A technology of human growth hormone and glycosylation, which is applied in the direction of growth hormone, hormone peptides, medical preparations containing active ingredients, etc.

Inactive Publication Date: 2011-07-20
NOVO NORDISK HEALTH CARE AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In general, lower than normal levels of hGH lead to growth-related defects

Method used

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  • N-glycosylated human growth hormone with prolonged circulatory half-life
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  • N-glycosylated human growth hormone with prolonged circulatory half-life

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Embodiment approach

[0125] CLAIMS 1. A human growth hormone variant, wherein said variant comprises an amino acid sequence comprising one or more N-glycosylation motifs (N-X-S / T) that are absent in wild-type human growth hormone.

[0126] 2. The human growth hormone variant according to embodiment 1, wherein at least one of said N-glycosylation motifs (N-X-S / T) absent in wild-type human growth hormone has been selected from the group consisting of Produced by mutations: S55N, Q69N, E74S, E74T, R77N, I83N, L93N, A98N, L101S, L101T, G104N, S106N, Y111S, Y111T, I121N, D130N, K140N, T142N, G161S, G161T, and E186N.

[0127] 3. The human growth hormone variant according to embodiment 1, wherein at least one of said N-glycosylation motifs (N-X-S / T) absent in wild-type human growth hormone has been selected from the group consisting of Generated by one or more mutations / mutation pairs: K41N, Q49N, S55N, E65T, E65T, E65N, Q69N, E74S, E74T, R77N, I83N, L93N, A98N, L101S, L101T, G104N, S106N, Y111S, Y111T, ...

Embodiment 1

[0260] Construction of vectors for expression of wild-type human growth hormone in mammalian cells

[0261] By means of flanking the sequence Hind III and Eco RI site, will Figure 1A The indicated nucleotide sequence was inserted into plasmid pEE14.4, resulting in plasmid pGB039. In pGB039, the nucleotide sequence encoding growth hormone was placed under the transcriptional control of a cytomegalovirus (CMV) promoter.

[0262] By inserting the pTT5 Hind III and not Between the I sites, the nucleotide sequence encoding growth hormone in pGB039 was subcloned, resulting in plasmid pTVL01.

Embodiment 2

[0264] Transient expression of wild-type human growth hormone in mammalian HEK293 cells

[0265] Suspension-adapted human embryonic kidney (HEK293F) cells (Freestyle, Invitrogen) were transfected with the pGB039 expression plasmid encoding wild-type human growth hormone according to the manufacturer's instructions. Briefly, 30 μg of plasmid was incubated with 40 μl of 293fectin (Invitrogen) for 20 min and added to 3 X 10 7 cell. in a shaking incubator (37°C, 8% CO 2 and 125 rpm) for 7 days. Media samples were collected daily and analyzed for human growth hormone using an ELISA kit (Roche).

[0266] The result of ELISA is as follows figure 2 shown, and demonstrated that transiently transfected mammalian cells are efficient producers of human growth hormone. The media harvested 7 days after transfection and dilutions of purified recombinant human growth hormone produced in bacteria were loaded onto SDS-PAGE gels and electrophoresed. Gels were stained with SimpleBlue Saf...

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Abstract

The present invention relates to novel human growth hormone (h GH) variant(s) with one or more N-glycans. The hGH variants of the invention comprises an amino acid sequence which includes at least one N-glycosylation motif (N-X-S / T) arising from one or more mutations not present in the wild type hGH. The h GH variants of the invention have a prolonged circulatory half-life and thus can be effectively used as a protein therapeutic for disease states that will benefit from increased levels of h GH. The process of obtaining the hGH variants is also encompassed by the invention.

Description

technical field [0001] The present invention relates to novel human growth hormone (hGH) variants having at least one N-glycosylation motif (N-X-S / T) which is absent in wild-type hGH. The hGH variants of the invention have prolonged circulating half-lives and are useful as protein therapeutics for disease states that would benefit from increased hGH levels. Background technique [0002] Human growth hormone (hGH) is a protein with 2 disulfide bridges, a length of 191 amino acids, and a molecular weight of 22 kDa. Disulfide bridges connect positions 53 and 165 and positions 182 and 189. hGH plays a key role in promoting growth, maintaining normal body composition, anabolism, and lipid metabolism (Barnels K, Keller U. Clin. Endocrinol. Metab. 10 , 337 (1996)). It also has direct effects on intermediary metabolism such as decreased glucose uptake, increased lipolysis, increased amino acid uptake and protein synthesis. The hormone also acts on other tissues, including adipos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/61C12N15/18C12N15/63C12N15/79A61K38/27A61P5/06
CPCC07K14/61A61K38/00C07K2319/91A61P5/06
Inventor G.博尔特C.克里斯滕森E.博尔T.V.伦德加尔德
Owner NOVO NORDISK HEALTH CARE AG
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