Expression, Purification And Activities Of Tα1-TP5 Fusion Peptide

Thymopentin(TP5),corresponding to the position 32-36 of thymopoietin,which exhibits similarly biological activities as thymopoietin,can have a function of two-ways regulation on imbalanced immune system.As an important immunomodulator,TP5 can induce T cell differentiation and promote the development and activation of T-lymphocyte subsets,resulting in curing immune deficiency and autoimmune disorders in clinical application.Because its half-life is very short(30 s),many TP5 analogs and peptidomimetics such as the insertion of stable components,D-amino acid substitution or cyclic peptide,have been prepared to enhance their half-lives and activities.However,all the results were still not satisfactory.Thymosonα1(Tα1),a natural polypeptide existing in a variety of biological tissues,is composed of 28 amino acid residues with a molecular weight of 3100 Da, pI 4.2,and has similar immunocompetence as TP5.As an immunomodulator,Tα1 has been used clinically for the treatment of immune defects.Plasm half-life of Tα1 was about 1.5 h.Currently,Tα1 is prepared from the animal tissues or chemical synthesis, so it has many shortcomings,e.g.low yields,and high price.Based on the facts that the proper extension of peptide chain can improve half-lives of peptide drugs,to elongate the half-life and activity of TP5,Tα1 was fused with TP5 and Tα1-TP5 fusion peptide was prepared by genetic engineering.1 Cloning and expression of Tα1-TP5 gene in P.pastorisIn this dissertation,a synthetic gene encoding a Tα1-TP5 was synthesized on the basis of its amino acid sequence and P.pastoris preferred codons.Upstream and downstream primers were designed by primer software,and then Tα1-TP5 gene was manipulated by PCR amplification,restriction enzyme digestion and ligation,and cloned into the P.pastoris expression vector pGAPZαA.The recombinant plasmid pGAPZαA-Tα1-TP5 was transformed into E.coli JM109,and then colony screening, restriction enzyme digestion,PCR reaction and sequence analysis showed that the synthesized gene and cloned gene were consistent completely with designed gene.There are some cases where unnecessary amino acids are left on the N- and C-terminus of the expressed fusion peptide.In our study,we inserted target gene rightly behind the Glu-Ala-Glu-Ala ofα-factor signal sequence,added a thrombin site gene between the C-terminus and stop condon,remaining 6×his tag.In this way, fusion peptide could be easily purified by metal chelating affinity chromatography, and the purified products could be further cleaved by thrombin to obtain Tα1-TP5 fusion peptide.BlnⅠ-lineared pGAPZαA-Tα1-TP5 was transformed into P.pastoris GS115 strain and positive colonies were screened with high concentration of Zeocin.Yeast genomic DNA was extracted by boiling-freezing-boiling and identified by PCR amplification.The recombinant transformants were cultured in the shake flask. Tricine-SDS-PAGE showed the production of Tα1-TP5 and found two transformants had high yields.The influence of culture time and temperature were studied.When cultured at 30℃to optical density at 72 h,the recombinant Tα1-TP5 fusion peptide reached 32.2 mg/L.2 Separation and purification of Tα1-TP5 fusion peptideThe technology of separation and purification of the fusion peptide was studied. After gaining the supematant by centrifugation,the macromolecules including proteases were removed by ultrafiltration,the fusion peptide was separated by metal chelate chromatography,and desalted and purified by G-25 gel filtration.Fusion peptide was digested by thrombin and was concentrated again.The purified Tα1-TP5 fusion peptide sample showed a single band by Tricine-SDS-PAGE.The correct molecular weight of Tα1-TP5 was further confirmed with ESI-MS.3 The secondary structure study of Tα1-TP5 fusion peptideThe secondary structure of Tα1-TP5 fusion peptide was determined by circular dichroism(CD) spectroscopy and Fourier transform infrared(FTIR) spectroscopy. The CD results showed that the secondary structure of Tα1-TP5 fusion peptide constituted of 32.01%α-helix,8.50%β-antiparallel,8.86%β-parallel,17.02%β-tum, and 33.61%random coil.Theα-helix and random coil covered the most part of the Tα1-TP5 fusion peptide,which was consistent with tendency of the Fourier transform infrared(FTIR) spectroscopy.4 In vitro plasma half life of Tα1-TP5 fusion peptideThe in vitro plasma half-life of each target peptide was determined by reversed-phase high-performance liquid chromatography(RP-HPLC) following incubation at 37℃in heparinized rabbit plasma at different time points.The results showed that the in vitro half-life of Tα1-TP5 fusion peptide,Tα1 and TP5 were (140±14) min,(127±11) min and(5.6±0.7) min respectively.The half-life of Tα1-TP5 fusion peptide is obviously longer than that of TP5 and Tα1 alone.5 Activities of Tα1-TP5 fusion peptideMouse spleen cell proliferation experiment showed that when the concentration was in the range of 10^-11 to 10^-6mol/L,the largest proliferation ratio of Tα1-TP5,Tα1 and TP5 were(50.97±5.19)%,(45.38±8.257)%and(40.61±5.17)%respectively. Tα1-TP5 has significantly higher activity(P＜0.01) than TP5 in promoting the proliferation of mouse splenocytes in vitro,but the activity difference between Tα1-TP5 and Tα1 was not significant.The in vivo macrophage phagocytosis experiments showed that the clearance index K of Tα1-TP5 fusion peptide,Tα1,TP5 and control were 0.020±0.0010, 0.016±0.0007,0.013±0.0008 and 0.005±0.0006 respectively.And their phagocytic index corresponded 3.95±0.19,3.73±0.56,3.43±0.29 and 2.48±0.40.All the peptide drugs could enhance the normal mouse macrophage phagocytosis,and Tα1-TP5 had better activity in promoting the phagocytosis of macrophages than TP5.The quantitative analysis of IL-2 in the serum of the peptide drug treated mice showed that the average IL-2 level of Tα1-TP5,Tα1,TP5 and control group were 848.81±46.70 pg/mL,761.50±31.33 pg/mL,749.62±29.08 pg/mL and 718.35±32.16 pg/mL respectively.These demonstrated that Tα1-TP5 fusion peptide has significantly higher activity in promoting T lymphocytes to secrete IL-2 than Tα1 or TP5 alone does.In view of the relations betweem structure and function of Tα1-TP5,enhanced activity may attribute to the small conformational changes of Tα1-TP5,compared with Tα1,especially the small increase ofα-helix structure,because in previous studies,it was presumed thatα-helix structure played a main role in the activity of Tα1.In summary,this study showed Tα1-TP5 might be used as a potential therapeutic agent for the human aliments.In this study,the main research results are as follows:(1) The vector pGAPZαA-Tα1-TP5 was constructed.Tα1-TP5 fusion peptide was expressed in P.pastoris and its expression level was detected to be 32.2 mg/L in the culture.ESI-MS confirmed its correctness of its structure.(2) A method of separation and purification of Tα1-TP5 fusion peptide in laboratory level was establishe.Tα1-TP5 fusion peptide was well purified by a serial of ultrafiltration,affinity adsorbtion and gel filtration and the purity of the product was over 99%.(3) The secondary structure of Tα1-TP5 was studied by CD spectroscopy and FTIR pectroscopy.Theα-helix and random coil covered the most part of its secondary structure.Further speculated theα-helix conformation was an important factor for its immunocompetence.(4) The in vitro plasma half-life of Tα1-TP5 was studied,and the results conformed that its half-life(140±14 min) is significantly longer than that of TP5 (5.6±0.7 min) or Tα1(127±11min).(5) The in vitro activity assay presented that Tα1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes,and in vivo experiment showed that Tα1-TP5 fusion peptide had better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Tα1 and TP5.Our findings suggested that Tα1-TP5 fusion peptide might be a potential therapeutic agent.In a word,this study has provided a basis for the further medicinal development of Tα1-TP5 fusion peptide.