Construction And Expression Of Recombinant Bovine Lactoferrin N-lobe In Pichia Pastoris

Lactoferrin(Lf) is an iron-binding glycoprotein which is rich in mammalian milk. It is currently found that Lactoferrin and its hydrolysis products have a diverse range of physiological functions, including broad spectrum antibacterial, antioxidation, antiviral, regulating immune response, as well as promoting iron absorption and so on. It is also considered as one of the most important composition in the mammalian nonspecific immune defense system.This study realized the heterologous expression of bovine lactoferrin N-lobe(1-333AA) in the Pichia pastoris expression system by means of construction the eukaryotic expression vector. The functional activities were also determined. Screening the positive strain was grown in a 5L fermenter to scale up fermentation, in order to provide practical basis and theoretical guidance for deeply study the structure and function of bovine lactoferrin, as well as further large-scale industrial production.The bovine lactoferrin N-lobe modified through codon optimization was synthesized based on maintaining the native amino acid coding sequence, considering the GC concent and the secondary structure near the AUG start codon, eliminating the mRNA degradation signal and the internal premature termination, splicing and replacing its own signal peptide sequence with theα-factor which is from Saccaromyces cerevisiae in pPIC9K vector. After optimized by the preference of the Pichia pastoris codon, 253 nucleotides were changed. The G+C content was adjusted from 56.39% to 40.42%. The secondary structure nearby AUG start codon was also optimized, in order to maintain the stability of mRNA, perform the translation successfully and promote the production of target protein.The Pichia pastoris expression vector pPICPK-BLfN-6His was constructed and linearized by Pme I , and then transformed and integrated in the genome of P.pastoris GS115 by electroporation. The recombinant protein was induced and expressed under the condition of 0.5% methanol at the shake level, and also deglycosylated using Endo H. SDS-PAGE and Western blot identified that the recombinant protein was successfully expressed and obtained correct glycosylation modification. The molecular weight of the rBLfN was 38kDa. The pH and dissolved oxygen were optimized. The higher expression recombinant was screened at shake level and fermentated in a 5L fermenter on the basis of optimized carbon source, pH and feeding strategy. After induced 96h, the secreted proteins concentration in supernatant was 540.07 mg/L.The recombinant protein was purified by His-bind Ni2+-affinity chromatography, and determined its biological function. In the iron-binding activity assay, the rBLfN showed a pH-dependent iron release. The iron-binding capability of rBLfN reduced gradually with the pH drops. In vitro antibacterial assay, compared with negative control (1.12×107 CFUs/ml), The MIC of 6mg/ml and 3 mg/ml against Staphylococcus aureus ATCC 25923 was 0.68×107 CFUs/ml and 0.92×107 CFUs/ml, respectively.