title: Molecular cloning, expression, and characterization of a Sophora alopecuroides lectin from Escherichia coli.
abstract: Sophora alopecuroides lectin (SAL) has been isolated from the seeds and confirmed to have antifungal and antitumor activities, and presently the preparation of the natural lectin was cumbersome, time-consuming, and the yield was relatively low for further analysis. In this study, the signal peptide of lectin, the modification sites, and the secondary structure were analyzed, and the three-dimensional structures of SAL were modeled. The gene of SAL was amplified by the reverse transcription polymerase chain reaction, and cloned into the pET-30a vector and expressed in Escherichia coli BL21(DE3) by the induction of isopropyl-beta-d-thiogalactopyranoside. Totally, 400 mg of recombinant SAL (rSAL) was purified from 1 l of bacterial culture through Ni-NTA agarose column and the purity reached 95%. The recombinant protein was further confirmed by western blot using rSAL-specific antibody. The biological activity analysis results showed that rSAL exclusively bound to d-galactose and had universal hemagglutinating activities to human A, B, O, and AB, and rabbit and mouse erythrocytes. rSAL also inhibited the growth of fungi, the proliferation of cancer cells, and the HIV-I reverse transcriptase activity. In conclusion, this study indicates that rSAL can be produced in large quantities in the prokaryotic expression system and the recombinant protein still retains the various biological activities, which will make the large-scale production of SAL recombinant protein at dramatically reduced cost possible.
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Jinyao
authors.initials: J
authors.lastname: Li
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Dongliang
authors.initials: D
authors.lastname: Liu
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China Texas Children's Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yang
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China.
authors.forename: Fuchun
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China sr_sun2005@163.com.
authors.forename: Surong
authors.initials: S
authors.lastname: Sun
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D029909:Sophora / Q000737:chemistry*; D013696:Temperature
chemical_list: D017931:DNA Primers; D037102:Lectins
keywords: SAL cloning; biological activities; prokaryotic expression; purification