A Mutant Recombinant Human Interleukin-2 Cloning, Expression, And Purification In Pichia Pastoris

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes.IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. Objective: Nearly these years, although genetically engineered IL-2 has been focused all of us attention upon its treatment of liver inflammation and several types of cancer, there are still some toxic side effects on sick men, such as heating, shivering with cold, nausea, feeling nauseated, fainting, expriencing exhausted and Capillary leakage syndrome when this drug was used with a large amount or for a long time.So it is necessary to reconstruct rhIL-2 for improvement of its stability and bioactivity, decreasing its toxic side effects. In order to obtain a high level express of human IL-2 in pichia pastoris, we modified the human IL-2 cDNA with PCR based mutagenesis method. And also we had determined the new mutant variant human IL-2 cDNA structure and its protein products activity. This method can be used to study gene modification, the structure or function of protein and the relationship between themselves. Methods: Mature human Interleukin-2 gene was amplified from IL-2 cDNA by PCR methods. By using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer, we replaced the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine. Restriction enzyme analysis was carried out and nucleotide sequences of the PCR products were determined by DNA sequence. Natural human IL-2 cDNA (NhIL-2 cDNA) and mutant variant human IL-2 cDNA(MvhIL-2) have been inserted downstream of promoter with pPIC9K expression vector, The PCR product precisely engineered into an intermidiate vector pPIC9K which digested with SnaBI, EcoRI, ligated by T4 DNA ligase and phosphatase alkaline. In order to obtain a lot of pPIC9K-MvIL-2 DNA, we transformed it into E.coli Top10F', expanding plasmids, then transfered into Pichia pastoris, and expressed the mutant protein(C125A/L18M/L19S) in Pichia pastoris. The biological activity of thepurified protein (IL-2- C125A/L18M/L19S and wild IL-2) were determined with MTT assay using IL-2-dependent CTLL-2 cells. Results: The mutant IL-2 was successfully constructed and expressed in Pichia pastoris. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E.coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and it has the added advantages of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia very useful as a protein expression system. Linearing pPIC9K-MvIL-2DNA with SalI, we transformed it into eukaryotic expression system?Pichia yeast. Transformants were fermented in a shaking incubator with baffled flask, analyzing the supernatants for protein expression by Coomassie-stained 15% SDS-PAGE, there are expressions of MvIL-2. It has immunocompetence by Western-blotting, we can get purified MvIL-2 by millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography. Purified Il-2-C125A/L18M/L19S protein had stimulating activity similar to...