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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.
Highlights
Human growth hormone is a 191-amino-acid peptide hormone secreted by cells of the human anterior pituitary
The pupa and silkworm cell line BmN, originally purchased from Invitrogen, have been propagated and stored in our laboratory, human erythroleukemia K562 cells were a gift from the Institute of Hematology of Chinese Academy of Medical Sciences [12], standard Human growth hormone (hGH) was purchased from Shanghai United Cell Biotechnology Co., Ltd., Plus Immobilization Kit was purchased from Thermo Corporation, mouse anti-hGH monoclonal antibodies were purchased from R & D Company, two-dimensional electrophoresis reagents and Superdex 200 prepacked columns were purchased from GE Healthcare, serum-free medium was purchased from Gibco, fetal bovine serum and RPMI1640 medium were purchased from Invitrogen, horseradish peroxidase (HRP)-conjugated goat
The results showed that BmhGH-like protein could bind to hGH antibody in both denatured and natural states
Summary
Human growth hormone (hGH) is a 191-amino-acid peptide hormone secreted by cells of the human anterior pituitary It is a nonglycosylated hydrophilic single chain globulin with a relative molecular weight of approximately 22 kDa [1]. It has extensive and important physiological functions, including promotion of growth and development of most tissues (except nervous tissue), organs, and the whole body; regulation of the human body’s metabolism; and various other physiological functions. The hGH can affect the proliferation and differentiation of most tissues and organs including bone tissue, cartilage tissue, adipose tissue, brain tissue, the immune system, the reproductive system, and the hematopoietic system [2] It can regulate various metabolic processes and can be used in the treatment of certain diseases. The exploration of hormone analogues to reduce these side effects is currently one of the hotspots of investigation of new hormone-like protein drugs
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