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Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk.

ABSTRACT: This study aimed to establish and reproduce transgenic pigs expressing human growth hormone (hGH) in their milk. We also aimed to purify hGH from the milk, to characterize the purified protein, and to assess the potential of our model for mass production of therapeutic proteins using transgenic techniques. Using ~15.5 L transgenic pig milk, we obtained proteins with ? 99% purity after three pre-treatments and five column chromatography steps. To confirm the biosimilarity of our milk-derived purified recombinant hGH (CGH942) with commercially available somatropin (Genotropin), we performed spectroscopy, structural, and biological analyses. We observed no difference between the purified protein and Genotropin samples. Furthermore, rat models were used to assess growth promotion potential. Our results indicate that CGH942 promotes growth, by increasing bone development and body weight. Toxicity assessments revealed no abnormal findings after 4 weeks of continuous administration and 2 weeks of recovery. The no-observed-adverse-effect level for both males and females was determined to be 0.6 mg/kg/day. Thus, no toxicological differences were observed between commercially available somatropin and CGH942 obtained from transgenic pig milk. In conclusion, we describe a transgenic technique using pigs, providing a new platform to produce human therapeutic proteins.

SUBMITTER: Lee SY

PROVIDER: S-EPMC7394428 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk.

Lee So-Young SY Han Joo-Hee JH Lee Eun-Kyeong EK Kim Young Kyu YK Hwang Seo-Ah SA Lee Sung-Hyun SH Kim Maria M Cho Gye Yoon GY Hwang Jae-Ha JH Kim Su-Jin SJ Yoo Jae-Gyu JG Cho Seong-Keun SK Lee Kyung-Ju KJ Cho Weon-Ki WK

PloS one 20200731 7

This study aimed to establish and reproduce transgenic pigs expressing human growth hormone (hGH) in their milk. We also aimed to purify hGH from the milk, to characterize the purified protein, and to assess the potential of our model for mass production of therapeutic proteins using transgenic techniques. Using ~15.5 L transgenic pig milk, we obtained proteins with ≥ 99% purity after three pre-treatments and five column chromatography steps. To confirm the biosimilarity of our milk-derived puri ...[more]

PMID: 32735629

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Project description:IntroductionGrowth hormone (GH) secretion and release is a complex and highly regulated process. Any alteration disturbing synthesis, secretion or biological action of GH, results into growth hormone deficiency (GHD). GHD is of two types-isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD), of which IGHDis more common. The genes implicated in its etiology are growth hormone 1(GH1) and receptor of growth hormone-releasing hormone (GHRHR). Mutations within the coding region and/or either entire or partial deletions of the GH1gene lead to IGHD. In addition, GH1 possesses upstream regulatory elements and a promoter with binding sites for various transcription factors, which control its expression.AimThe study was planned with an aim to identify entire GH1 locus deletion, mutations in the GH1 coding region and sequence variations (polymorphisms) in the promoter region of the gene in patients with IGHD.Materials and methodsThirty patients clinically diagnosed with IGHD and 30 healthy individuals who formed the controls were enrolled for the study. Genomic DNA was isolated from peripheral blood sample and processed for amplification of the desired regions followed by direct sequencing and/or restriction endonuclease digestion.ResultsOut of the 30 IGHD patients screened, 20% of the cases showed consanguinity and 16% had a positive family history. Seven percentage of the patients showed homozygous deletion of the GH1gene while rest of them had heterozygous deletion. Screening of the coding region of GH1 showed sequence variations in exon 1 in 20% of the patients whereas the promoter region showed the presence of polymorphisms-rs2005171 in 20%, rs2005172 in 15% and rs11568828 in 18% of the cases. The haplotype comprising rs2005171 and rs2005172 was observed in four patients.ConclusionThe present study is an attempt to characterize the GH1 locus in IGHD patients. To the best of our knowledge this is the first study of its kind where entire GH1locus, upstream regulatory elements and promoter region have been studied. Such an analysis would provide valuable information on the etiology of IGHD.

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Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk.

Publications

Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk.

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