title: Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.
abstract: Mouse NGF (mNGF) extracted from mouse submaxillary gland has been approved on the market in China for treating nerve damage caused by N-hexane poisoning for over a decade, and many researches showed the clinical effectiveness of mNGF for the treatment of other nerve system diseases. The extracted mNGF have risks of potential viral contamination due to the animal origin. Here, we report the successful expression, purification, and characterization of recombinant mNGF (rmNGF). An expression plasmid of mouse nerve growth factor (mNGF) was constructed and transfected into CHO-S cells. Stable transfectants were obtained using a two-phase selection scheme with the addition of different concentrations of methotrexate and puromycin. Recombinant mNGF (rmNGF) was purified from cell culture medium by a two-step procedure: cation exchange followed by size-exclusion chromatography. The purity of rmNGF was 98.6% determined by size exclusion high performance liquid chromatography (SEC-HPLC). The molecular weight, isoelectric point and N-terminal sequence of rmNGF were identical to the theoretical values entirely. In TF-1/MTS, the specific activity of the protein was approximately 1.7×10(6)U/mg against rhNGF (the reference standard). In DRGs, the specific activity was approximately 7.3×10(5)AU/mg against mNGF (the reference standard). Our results showed that a high quality of rmNGF with marked biological activity comparable with mNGF was produced, and laid the basis for further research and development of rmNGF.
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Xinchang
authors.initials: X
authors.lastname: Shi
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Chunmei
authors.initials: C
authors.lastname: Han
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Tao
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing, PR China. Electronic address: raocm@nicpbp.org.cn.
authors.forename: Chunming
authors.initials: C
authors.lastname: Rao
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D049109:Cell Proliferation; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D020932:Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D020932:Nerve Growth Factor
keywords: Characterization; Expression; Nerve growth factor