Polycistronic Expression Of Human Platelet Factor4in Escherichia Coli

Platelet factor4（PF4）, stored in α-granules, is a CXC-chemokine released uponplatelet activation. In addition to binding to and neutralizing heparin, PF4has severalbiological activities, including chemotactic activity for human monocytes andneutrophils, involvement in fibroblast adhesion, immunoregulatory activity andinhibition of tumor growth. Its relative molecular weight is small, so it is susceptibleto rapid degradation in vivo and its half-life is very short. So that expression andpurication of PF4often encountered many difficulties, which caused industrialproduction to be restricted to some extent. In this paper, polycistronic expression ofthe tandem repeat gene was used to express human PF4, which was a novel way toachieve large-scale production of PF4protein for possible researches and clinicalapplications.To begin with, primers were designed according to the optimized PF4geneswhich were amplified by overlapping PCR. And the amplification products weresubcloned into the clone vector pEASY-T3. Then the series unit, first series unit andcorresponding primers were designed and amplified by overlapping PCR. And theamplification products were respectively subcloned into the clone vector pEASY-T1to generate pEASY-T1-PF4and pEASY-T1-f PF4. Furthermore, plasmidspEASY-T1-PF4and pEASY-T1-f PF4were digested by isoaudamers to get large andsmall fragments, which were ligated by T4DNA ligase to generate pEASY-T1-2f PF4.So that plasmids pEASY-T1-nf PF4（n=1,2…6） were acquired finally according to the same method described above. Finally, the large fragments of expression vectorpBV220and pET-28a（+） were recycled after digested by isoaudamers. An n×PF4DNA fragment （n=1,2,3,4,5,6） was acquired from the clone vector pEASY-T1-nfPF4（n=1,2…6） digested by isoaudamers, and then ligated by T4DNA ligase with theDNA fragments from pBV220and pET-28a（+） to generate the expression vectorspBV220-nf PF4and pET28a（+）-nf PF4（n=1,2…6）. The confirmed recombinantvectors were respectively transformed into E. coli DH5α and BL21（DE3） cells tocreate E. coli DH5α/pBV220-nf PF4and E. coli BL21（DE3）/pET28a（+）-nf PF4（n=1,2…6）. The fusion protein His-PF4wasn’t or was little detected in the proteins fromwhole bacterial cells DH5α/pBV220-nf PF4（n=1,2…6）; The strain E. coli BL21（DE3）/pET28a（+）-nfPF4（n=1,2…6） was induced in the same condition(OD₆₀₀=0.6±0.02,0.6mmol/L IPTG,12h) and the His-PF4fusion protein was detected inthe supernatant as a soluble protein, as an insoluble protein inclusion body in the totalcell protein extract after ultrasonication. SDS-PAGE and Western blot of wholebacterial proteins showed that the expression of the target protein increases graduallyalong with growing copy number. When copy number is four or more, proteinexpression does not increase significantly. Scan analysis showed expression levels ofabout44.45%,44.03%, and44.28%of total proteins in E. coli BL21（DE3）/nf PF4（n=4,5,6）. Engineering bacteria BL21（DE3）/pET28a（+）-4f PF4was induced by IPTG.The high-purity （above95%） recombinant protein could be obtained once by affinitychromatography and gel filtration desalting column. The purified fusion protein wasfurther identified by cleavage with enterokinase and MS （Mass Spectrometry）. And itsactivity was determined using colony formation assay, which showed that purifiedHis-PF4protein could obviously inhibit colony formation of HEL （Erythroleukemiacell line） cells with inhibition rate of55.6%.This thesis tries to solve the technical problems related to recombinantpolypeptide drugs, such such as the low production, the difficulties associated withpurification process, and extra amino acid residues left at the N-or C-termini of thesmall proteins following cleavage. It provides an effective and alternative plan for thecurrent production process of PF4and a new way of biosynthesis of polypeptidedrugs.