Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.
We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.
Joshi
Bharat H
BH
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA.
Puri
Raj K
RK
D036002:ADP Ribose Transferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D005822:Genetic Vectors; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D018793:Interleukin-13 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D007544:Isopropyl Thiogalactoside / Q000031:analogs & derivatives* / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D009113:Muramidase / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use; D013997:Time Factors; D014170:Transformation, Genetic; D037521:Virulence Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000900:Anti-Bacterial Agents; D001120:Arginine; D001427:Bacterial Toxins; D005098:Exotoxins; D005973:Glutamine; D018793:Interleukin-13; D007544:Isopropyl Thiogalactoside; D007612:Kanamycin; D009113:Muramidase; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C070641:isopropyl-beta-galactopyranoside