| Pichia pastoris expression system is an eukaryotic expression system.The use of yeast system to express exogenous proteins has become more and more mature.Bovine lactoferrin has a wide range of biological activities,such as antibacterial,antiviral,antineoplastic and immune regulation.It has broad application prospects in food additives,feed additives,medicine and cosmetics industries.Because of the high cost of extracting lactoferrin from milk,it can not be widely used in industrial production.Secretory expression of bovine lactoferrin functional fragment in Pichia pastoris,Furthermore,the purification,identification and functional study of the target protein were carried out,and study the differences of their expression in different high-density fermentation media.The results of research are shown as follows:In this study,the N-terminal functional fragments of bovine lactoferrin（including 1-333 amino acid sequences of two functional domains,Bovine lactoferrin functional fragment,BlfFf）was electrotransformed into Pichia pastoris GS115.Positive transformants with different resistance were screened by histidine deficiency and G418 gradient resistance.To determine whether recombinant bovine lactoferrin functional fragment is expressed by SDS-PAGE and Western Blot identification.Genomes of transformants with different resistance were extracted,and 1,2,3and 4 copies of the strains were screened by double standard curve method of RT-PCR,After shaking flask fermentation and induction by methanol,The target proteins were quantitatively analyzed by ELISA kit.The results showed that the expression of four copies of the strain was high,the shaking flask yield reached 1.9mg/L,which was nearly 25%higher than that of single copy,The strain was named GS115-pPIC9K-BlfFf-10.Purification of fermentation-induced supernatant,The results showed that the combination of cation exchange chromatography and nickel affinity chromatography had the best effect.The recovery of target protein was 45.7%in cation exchange,54.0%in nickel affinity chromatography and 20.6%in the whole purification process.The electrophoretic purified functional fragment of bovine lactoferrin was successfully obtained,The bovine lactoferrin functional fragments were identified by LC-MS,The molecular weight and sequence of the identified fragments were consistent with the software prediction,The molecular weight and sequence of the fragment were consistent with the software prediction.The functional fragment of bovine lactoferrin after pepsinization had good antimicrobial activity against Staphylococcus aureus and Escherichia coli by bacteriostatic experiment,the antimicrobial rate reached 69.86%and 52.49%,respectively.The fermentation conditions of GS115-pPIC9K-BlfFf-10 strain were optimized at shaking flask level.The optimized fermentation conditions were as follows:initial pH 5.0 of medium,50 mL shaking liquid（500 mL conical bottle）,2.0%methanol added every 24 h,and induction temperature of 28 C.The effects of RDM,BSM and D’Anjou high density fermentation medium on the expression of target protein were compared by 5L fermentor.The results showed that after 72 h fermentation induction,The wet weight of the strain reached 380 g/L,342 g/L and 285 g/L,The expression of the target protein was 38.1 mg/L,29.52 mg/L and 20.25 mg/L and the average specific growth rate of the strain in the three media was 0.0128 h-1、0.0175 h-1 and 0.009 h-1.Therefore,Selecting RDM as high density fermentation medium can not only reduce the cost of medium,but also reduce the cost of desalination in the process of separation and purification,This study laid a foundation for potential for industrial applications. |
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