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Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm
Jianqing Chen1, Tejun Shu1, Zhengbing Lv1
1Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Abstract:
Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.
Insights
Researchers discovered a human growth hormone-like protein in silkworms. This protein, BmhGH-like, promotes cell division and may lead to new hormone-based drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Human growth hormone (hGH) regulates metabolic pathways.
- The anterior pituitary secretes hGH.
- Silkworms are a potential source of novel biomolecules.
Purpose of the Study:
- To identify and characterize a protein in silkworms with hGH-like properties.
- To investigate the cell division-promoting effects of this silkworm protein.
- To explore the potential medicinal applications of silkworm-derived hormones.
Main Methods:
- Western blotting and immunoprecipitation to detect the protein.
- Purification using chromatography and electrophoresis.
- Mass spectrometry (MS MALDI-TOF-MS) for protein analysis.
- Cell-based assays (K562 cells) and flow cytometry to assess cell division.
- Recombinant baculovirus expression system for functional studies.
Main Results:
- A 30-kD protein, BmhGH-like, was identified in silkworm pupae.
- BmhGH-like protein consists of two subunits, hGH-L1 and hGH-L2, belonging to the β-trefoil superfamily.
- The protein's structure is similar to hGH.
- In vitro assays showed the protein promotes K562 cell division.
- Recombinant hGH-L2 shortened the G0/G1 phase and promoted cell cycle progression in BmN cells.
Conclusions:
- Silkworms possess a novel hGH-like protein.
- This protein exhibits growth-promoting activity by enhancing cell division.
- The discovery opens avenues for developing new hormone-based therapeutics from silkworms.
