Purification And Characterization Of Recombinant Human Interleukin 11 Expressed By Pichia Pastoris

Chemotherapy induced thrombocytopenia can be treated by recombinant human interleukin eleven( rhIL-11). Commercial available rhIL-11 is produced by the traditional method with E.coli engineering bacteria expressing rhIL-11 as a fusion protein. Its downstream purification processing is very complicated . Recently developed Pichia pastoris expression system , is easy to operate with low cost. We synthesized the human IL-11 gene according the sequence after optimized by computer and constructed the Pichia pastoris rhIL-11 expression strain. With this strain, the downstream purification processing technique was investigated and characterization of the purified rhIL-11 was carried out. The engineering strain was cultured with glycerol media in 15 liters automatic fermentation tank. Methanol was then added to induce the expression of rhIL-11 after glycerol was exhausted. The amount of rhIL-11 produced by the engineering yeast is greater than 0.4g/L. The supernatant was collected by centrifugation, and then concentrated by tangential flow ultra-filtration system with 5KD NMWL membrane to remove the substances of small molecular weight. After ultra-filtration, the conductance of the supernatant is less than 5mS/cm.For selecting cation ion exchange media for initial purification, the separation efficiency of CM-Sepharose Fast Flow and SP-Sepharose Fast Flow was compared. It was found that their separation efficiency was identical, while the former gave a better recovery of yield. We also studied the elution condition of cation ion exchange chromatography. The final optimized condition was: after loading of the sample, the column was washed with buffer A(20mmol/L Tris-HCl pH8.5, 5mmol/L EDTA) to baseline, and then elute with 2 x column volumeof 15% buffer B(buffer A+500mmol/L NaCl). Finally it was eluted with 30% buffer B and the target protein was collected .Selecting Phenyl-Sepharose HP ( 2.6/20cm) as media for hydrophobic interaction chromatography(HIC), the column was equilibrated by 1.6mol/L (NH4)2SO4 -lOmmol/L Tris-HCl, pH8.5. (NH4)2SO4 concentration of the target peak after cation ion exchange was adjusted to 1.6mol/L by adding 3.2mol/L (NH4)2SO4 solution. After loading this sample to HIC column, the column was washed with 1.6mol/L (NH4)2SO4- lOmmol/L Tris-HCl(pH8.5) to baseline . The column was then eluted with 3 x column volume of 10% eluting buffer(10mmol/L Tris-HCl, pH8.5), and the target peak was collected when increasing elution buffer to 40% within 2 x column volume. At last obtained was the purified rhIL-11 after applying the target peak of HIC to desalting column桽ephadex G-25.The characteristics of purified rhIL-11 thus obtained was analyzed and compared with NEUMEGA which is a commercially available product expressed in E.coli with fusion expression system. The following analytic methods were conducted: SDS-PAGE; Western-blotting; IEF; RP-HPLC[C4 column(15cmx4.6mm,Vydac), the mobile phase consisted of solvent A(0.1% TFA in 90/10(V/V) acetonitrile/water) and solvent B (0.1% TFA in 90/10(V/V) acetonitrile/water) .The separation was performed by a liner gradient of 25%-80% over 30 min at a flow rate of O.Sml/minand detected by UV absorbance at 214nm];SEC-HPLC [performed with Bio-Sil SEC 250-5(Bio-Rad),the mobile phase is 0.05mol/L phosphate buffer(pH6.8) - 0.15mol/L sodium chloride at a flow rate of 0.8ml/min.Detected by UV absorbance at 280nm];peptide mapping with trypsin degradation; mass spectrometer,N and C terminal amino acid sequencing and bioactivity. The results proved that the rhIL-11 expressed by Pichia pastoris was just same as NEUMEG. The purity is greater than 97% and the specific activity is 8.2X 106Tj/mg.So the method established in this study is possibly a more simple and less expensive one to produce rhIL-11 for clinical use.