The Research Of Recombinant Expression And Stability Of LZ8-Fc Fusion Protein

Ganoderma lucidum (G. lucidum) has been used in traditional Chinese medicineherbs for countries, and it is regarded as effective nutrients for human bodies. Modernmedical studies have confirmed that G. lucidum extracts have anti-tumor andimmune-regulatory functions. However, most of the studies focused on the mainactive components of G. lucidum such as polysaccharides and triterpenoids. In1989,Kino and colleagues extracted and purified a fraction of small molecule proteins fromthe mycelia of G. lucidum, and named it Ling Zhi immune-regulatory protein (LZ-8).This discovery confirmed the existence of immunomodulatory proteins. Ever sincethen, dozens of research groups investigated in-depth studies on LZ-8through variousgenetic expression systems to obtain the recombinant LZ-8. The crystal structure ofLZ-8has been revealed and the main physico-chemical properties are known. Mostimportantly, the therapeutic value of immunomodulation and antitumor activities ofLZ-8have been studied. Nevertheless, the pharmaceutical development based onLZ-8has not been favorable mainly due to the small molecular weight of LZ-8(13kDa) and its poor stability in vivo. In addition, LZ-8belongs to heterologousprotein family derived from G. lucidum, which leads to serious immune rejection. Inthis study we fused LZ-8with human IgG Fc fraction to produce the LZ8-Fc fusionprotein in a yeast (pichia pastoris) expressing system. The fermentation andpurification technology of LZ8-Fc was established, and its stability in vivo waspreliminary studied. This study will benefit the pharmaceutical development of LZ-8.For technical approaches, the flexible peptide (GGSS) was used to connect LZ8and IgG1Fc fraction. The DNA sequence was optimized to yeast codons. The EcoRIand KpnI restrictive enzyme sites were inserted to both terminals of the LZ8-Fccoding sequence.The the LZ8-Fc coding sequence was inserted to the plasmid (pPICZɑA). AOX1promoter was also inserted in the vectorfor methanol secretory induction. Therecombinant plasmid was named pPICZɑA-LZ8-Fc and transformed to the yeast strain (X33). The preliminary methanol-inducible expression condition of LZ8-Fc wasoptimized..In the40-liter pilot-scale fermentation, the fermentation parameters, includingpH, DO, tank pressure, temperature, ventilation, were further optimized for methanolinduction of LZ8-Fc expression.The results of SDS-page electrophoresis and Western Blot analysis on thesupernatant of fermentation indicated that LZ8-Fc protein bands were detectableduring methanol induction, and the degradation bands of LZ8-Fc were detectableafterwards. The fermentation supernatant was treated by a two-stage tangential flowfiltration. Afterwards, the samples were purified by MabSelect Sure affinity andSuperdex75gel filtration chromatography. It was determined that the purificationprocess was steady, and high purity LZ8-Fc fusion proteins were obtained.The resultof electrophoresis showed a single band, and the result of mass spectrometry analysiswas expected. However, the Western Blot analysis indicated that the LZ8-Fc wasdegraded during purification. The LZ8-Fc fusion protein concentration was248g/mL,and the purity was98.33%, detected by high-performance liquid chromatographicidentification. The hemagglutinating activity examination showed that LZ8-Fc hadlower activity than LZ-8in sheep red blood cell agglutination. γ interferon activityof splenocytes was carried out in mice. The result indicated that LZ8-Fc had nostimulation activity of γ-interferon on spleen cells compared with LZ-8. The half-lifedetection was performed by tail vein injection. The blood was collected at5min,30min,1h,2H,4H,8h,24h,48h time points, and the results indicated that rLZ8concentration in serum decreased significantly at8h and out of detection at24h, whileLZ8-Fc was out of detection at4h.According to the information of SCOP online database, it showed that the crystalstructure of LZ-8and IgG Fc were both globulin. Thus, the retention time analysis ofLZ8-Fc was performed through molecular screen HPLC. The LZ8-Fc molecularassembly was a dimer. This study also found the phenomena that LZ8-Fc proteinstructure was broken in high concentration (2.5mg/mL). Molecular dynamics,combined with the structural analysis of circular dichroism spectra were utilized in thestability analysis of LZ8-Fc. It was discovered that considerable changes happened tothe secondary structure of LZ8-Fc after expression, which may explain the poor stability of LZ-8.This study was carried out to enhancing the stability of LZ-8in vivo and toreduce its immunogenicity as a heterologous protein. Although the LZ8-Fc protein isnot very stable, the results suggested it is important to improve the expression offusion protein. The downstream of the N-terminal of the protein may interrupt thefolding of LZ8-Fc. Therefore, the downstream protein was suggested to retain, whichprovided a theoretical basis for further studies on fusion proteins.