The Cloning And Expression Of Murine Lactoferrin In Escherichia Coli And Pichia Pastoris

Lactoferrin(LF) is an iron-binding glycoprotein of the transferrin family.It is widely found in milk,saliva,tears and other secretions.Lactoferrin is an important host defense molecule and has a diverse range of physiological functions,including antimicrobial,antiviral,and antioxidant activities,as well as immune regulation,modulation of cell growth and transcriptional activation of several genes.Lactoferrin has been one of the hot spots of research all over the world since it was found.In the present work,Mus musculus lactoferrin cDNA was cloned for the construction of prokaryotic and eukaryotic expression vectors,which then were expressed in Escherichia coli and Pichia pastoris expression system,respectively.The biological activities of the recombinant proteins were measured.This work will help the study of the structure and function of lactoferrin, as well as its further large scale production and application.Total RNA was isolated from the mammary gland of lactating Mus musculus.The lactoferrin cDNA was synthesized by reverse-transcriptase polymerase chain reaction(RT-PCR).Sequencing shows that the cDNA(2121 bp) encodes a precursor peptide chain consisting of 707 amino acids with a signal peptide of 19 amino acids.The sequence homology of the signal peptide and mature peptide between MLF and 10 other mammalian LFs is more than 50%;only six amino acids(Arg6, Met63,Ser359,Ala381,Glu420 and Ala615) in mature MLF are different from that of house mouse LF.The expression vectors pET28a-MLF and pET28a-MLFN were constructed and expressed in E.coli BL21(DE3).The products were examined by Western blot.The molecular weight of the MLF(murine lactoferrin) and MLFN(murine lactoferrin N-lobe) were 81.73 kDa and 38.72 kDa, respectively,and most of the products exsited in the inclusion bodies.17 mg MLF and 20 mg MLFN were obtained from 1 liter of E.coli culture after Ni²⁺-affinity chromatography under denatured condition.The yields of bioactive MLF and MLFN after refolding were 0.06 mg/L and 0.09 mg/L,respectively.In antibacterial activity assay,the viability of Streptococcus aureus (ATCC 25923) was modestly reduced by incubation of the cells with 25μmol/L recombinant MLFN(7.2×10⁶ CFUs/ml) compared with the negative control(1.4×10⁷ CFUs/ml).The recombinant MLF(12.5μmol/L) didn't display obvious antimicrobial activity against S.aureus.The coding sequence of lactoferrin N-lobe was subcloned into pichia expression vector pPICzaA to generate the expression vector pPICZaA-MLFN.pPICZaA-MLFN constructs containing two to eight copies of MLFN was also constructed.The pPICZaA-MLFN construct was transformed into Pichia pastoris X-33,and the foreign protein of 39.6 kDa was detected by SDS-PAGE and Western blot after 0.5%methanol induction.The expression level was highest after 48 h of induction.The yield of foreign proteins was 5 mg/L by Ni²⁺-affinity chromatography. Iron-binding assay of the recombinant protein showed that the iron release of the recombinant MLFN was pH-dependent.The iron-satureted level of recombinant lactoferrin N-Lobe decreased as the pH was lowered.In antibacterial activity assay,the viability of S.aureus was modestly reduced following incubation with 50μmol/L rMLFN(9.7×10⁶ CFUs/ml) compared to the negative control(1.4×10⁷ CFUs/ml),while rMLFN(25μmol/L) didn't display obvious antimicrobial activity against S.aureus.