Expression, Refolding, Purification And The Pilot-scale Production Study Of Recombinant Human Interleukin-1 Receptor Antagonist

With the development of biotechnology, large-scale production of recombinant protein is possible.Interleukin-1 receptor antagonist (IL-1ra), a naturally occurring anti-inflammatory protein, competitively blocks the binding of IL-1αand IL-1βto their receptors. High dosage of IL-1ra are needed to relieve disease because even very low IL-1 can induce complete biological response. The dosage is 1-2 mg/kg/d when treating RA. The aim of the present study was to establish a method for large-scale producing IL-1ra by recombinant technology.1. cDNA encoding of IL-1ra were synthesized, and the seed strain was constructed by transformation of PLY-hIL-1ra into host strain BL21, designated as BL21/PLY-4-hIL-1ra.2. Fermentation parameters were investigated in shaking bottle. Inducetion point was exponentional phase stage of growth. The target protein synthesis was induced at 42°C for 4h. Then, fermentation was established in a 3 L and 30 L bioreactor was trialed, respectivlely.In 30 L bioreactor, cell weight was 60g/L, and the rate of recombinant protein was 34%.3. The purification of rhIL-1ra was studiedi Rupturing cells and releasing inclusion body. High-pressure homogenization was used and the rate of cell rupture was 96%.ii Washing inclusion body. The rate and purification of target protein was 89.6 %and 72 %, respectively.iii Dissolving inclusion body. Dissolving buffer (ml): inclusion body (g) =20:1. Inclusion body is dissolved for 6h with stir (200 rpm). Recipe of dissolving buffer: 20 mM Tris-HCl, 5 mM EDTA, 5 mM DTT, 6M urea, pH8.0.4. Protein refolding with chromatography. Anion exchange chromatography column (Q-Sepharose Fast Flow) was used. The precodure was perfomed at 10°C, pH 8.0. The concentration of urea was reduced from 6 M to 2.1 M. The volume of refolding buffer was four fold of column volume and the linear velocity was 0.565 cm/min. Two hundred milliliter sample solution with 1024 mg target protein was loaded onto chromatography column and eluted with linear gradient elution with a rate of 6.0 ml/min. The concentration sodium chloride varied from 0 M to 0.4 M within 50 min. The recovery rate was 70 %. Specific activities was 100 000 U/mg. The purity was 98%.These parameters were applied to a large-scale production and excellent results obtained.5. Strong cation-exchange chromatography column (SP-Spharose Fast Flow) was used to remove endotoxin.With this technique did endotoxin flow through the column by adjusting pH but protein absorbed. The level of endotoxin was decreased to 0.5 EU/mg and the recovery rate was 93%.6. Storage condtion. rhIL-1ra should kept in 1.8 % sucrose solution at 20°C, pH6.5.7. Analysis of Specification. i. Molecular weight determined by MADLI-TOF MS was 17.25 kD. ii. Purity of original protein solution was not less than 98% by RP-HPLC. Specific bioactivity was 100 000 U/mg by thymus cell assay.