title: Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.
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.
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Jianqing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Tejun
authors.initials: T
authors.lastname: Shu
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Zhengbing
authors.initials: Z
authors.lastname: Lv
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Zuoming
authors.initials: Z
authors.lastname: Nie
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yu
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Qijing
authors.initials: Q
authors.lastname: Gai
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China; College of Pharmacy, Tianjin University, Tianjin 300073, China.
authors.forename: Yaozhou
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000737:chemistry*; D002846:Chromatography, Affinity; D019382:Human Growth Hormone / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D020014:K562 Cells; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011679:Pupa / Q000737:chemistry; D040681:Structural Homology, Protein
chemical_list: D019476:Insect Proteins; D019382:Human Growth Hormone