title: High-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide.
abstract: Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells.
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China.
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China.
authors.forename: Mingxin
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China.
authors.forename: Yimin
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China.
authors.forename: Liyuan
authors.initials: L
authors.lastname: Ge
authors.affiliation: Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Xu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China; Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China. Electronic address: caolin@njau.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, Jiangsu, PR China. Electronic address: dr.guohongqian@gmail.com.
authors.forename: Hongqian
authors.initials: H
authors.lastname: Guo
mesh_terms: D000209:Acute-Phase Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D053528:Lysobacter / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics
chemical_list: D000209:Acute-Phase Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D012709:Serum Albumin
keywords: Acute kidney injury (AKI); Mammalian cells; Neutrophil gelatinase-associated lipocalin (NGAL); Secretory expression; Signal peptide