Chemical Modification Of Recombinant Human Interleukin-2

Recombinant Human Interleukin-2(rhIL-2) is a more important lymphokine, possesses many of biological activities and phamacentical value. However, rhIL-2, purified from E.coli, has limited solubility at neutral pH and has a relatively short half life, heteroproteins may cause immunoreaction in vivo and decrease the effect of drug. rhIL-2 has a total of 133 amino acids including CYS58,CYS105 and CYS125, CYS58 and CYS105 formed disulfide bond, its molecular weight is 15420, pI is between 7.7-8.2,specific activity is 1x107IU/mg protein. Our research is about chemically modifying rhIL-2 with polyethylene glycol(PEG). PEG will be linked to rhIL-2, there will be a perfect protective screen after modification, thus rhIL-2 modified by PEG would not be recognized as the antigen in vivo and inhibit the coming immune reaction, lighten its decreasing rate in blood plasma, prolong its half life, increase its exposing time and decrease its unsteady waves in blood plasma. We chose PEG as the raw material to modify rhIL-2 because PEG was admitted to be used on human beings by the FDA. Though used Methoxypolyethylene Glycol activated with cyanuric chloride(MPEG-CC) and Methoxypolyethylene Glycol p-nitropHenyl carbonate(MPEG-NC) to modify rhIL-2, we drew the conclusion that rhIL-2 would be modified by an different active ester of PEG and be selected different condition. In our study, we discovered that MPEG-CC was easy to be hydrolyzed, but PEG-4000 could restrain it. By comparison, we selected MPEG-NC to modified rhIL-2. Based on the characterization of rhIL-2 and used SDS-PAGE, MTT colarimetry as the basical examing methods. We selected pH 9.3, 25℃, MPEG-NC vs rhIL-2 30:1(molar ratio) as the best condition and one hour later stopped by acetic acid. The proportion of the total production of modified rhIL-2 was up to 75%. The modified rhIL-2 was separated and purified from non-modified rhIL-2 by Phenyl Sepharose 6 Fast Flow and Superdex 75. The production with the purity of 95% and activity ratio was obtained. The total harvest rate of the protein was 23%. We detected remained PEG and found that it was suitable to use PEG-rhIL-2 on animal models.We measured the thermostability, the ability against Trypsin and solubility in water of PEG-rhIL-2, and the results showed that PEG-rhIL-2 was prior to rhIL-2.