High-Level Expression, Purification, and Renaturation of Recombinant Murine Interleukin-2 from Escherichia coli

Guisez, Yves; Demolder, Jan; Mertens, Nico; Raeymaekers, A.; Plaetinck, Geert; Robbens, Johan; Vandekerckhove, Joël; Remaut, Erik; Fiers, Walter

doi:10.1006/prep.1993.1031

Cited by 18 publications

(15 citation statements)

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“…Clearly bacterial challenge gives a far larger insult to the fish, and results in IL-2 up-regulation. Whilst the function and activity of IL-2 is well known in homeotherms, where the recombinant protein has been made and tested in many species including humans [40,41], mice [42], sheep [43,44], horses [45], pigs [46], chickens [47] and ducks [48], due to the uncertainty of the function of this molecule in fish, bioactivity studies are needed to confirm the role of IL-2 in the piscine immune response. Following successful production of the trout rIL-2 in this study the impact on the expression of seven different genes was studied: STAT5, Blimp-1, IL-1b1, IL-2, IFN-g1, COX-2 and gIP.…”

Section: Discussionmentioning

confidence: 98%

Rainbow trout interleukin-2: Cloning, expression and bioactivity analysis

Díaz‐Rosales

Bird

Wang

et al. 2009

Fish & Shellfish Immunology

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Section: Discussionmentioning

confidence: 98%

Rainbow trout interleukin-2: Cloning, expression and bioactivity analysis

Díaz‐Rosales

Bird

Wang

et al. 2009

Fish & Shellfish Immunology

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“…In our case, refolding of WbpO was optimal at approximately 30 h. It is also important to perform dilution and concentration steps slowly to minimize the precipitation of the target protein. Other refolding procedures, which involved sequential dialysis with decreased concentrations of urea (46,47) followed by renaturation of the protein immobilized to affinity resin (48) and renaturation of protein in 20% glycerol (49), generally result in very low yields because of protein precipitation problems. The procedure described in this paper provided an optimal condition to achieve high purity and high yield of WbpO with tight secondary structure.…”

Section: Discussionmentioning

confidence: 99%

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Zhao¹,

Creuzenet²,

Bélanger³

et al. 2000

Journal of Biological Chemistry

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WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-D-galactosamine (UDP-GalNAc) to UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO Sol ) exhibited a secondary structure with 29.2% ␣-helix and 20.1% ␤-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO Rf ) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO Rf catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD ؉ and NADP ؉ as the cofactors, respectively. The K m values of WbpO Rf for UDP-GalNAc, NAD ؉ , and NADP ؉ were 7.79, 0.65, and 0.44 mM, respectively. WbpO Rf can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD ؉ /NADP ؉ -dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

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“…The inclusion bodies were collected by centrifugation, solubilized in 20 mM Na 2 HPO 4 , 500 mM NaCl, 10 mM imidazole and 8 M urea, pH 7.4, and purified in a nickel-Sepharose affinity column (Amersham Biosciences, Uppsalla, Sweden), following the manufacturer’s recommendations. The purified protein was refolded using the dialysis method described by Guisez and collaborators [31] and the protein concentration was determined by Bradford’s method.…”

Section: Methodsmentioning

confidence: 99%

“…His-tagged recombinant green fluorescent protein (EGFP), produced in BL21(DE3)pLysS E. coli , and chromatographically affinity purified, was used as a positive control. To promote stability of the purified protein, bovine serum albumin was added to achieve a final concentration of 1 mg/mL [31]. Aliquots were prepared and stored at -20°C until use.…”

Section: Methodsmentioning

confidence: 99%

Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells

et al. 2014

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BackgroundVery few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs.ResultsRecombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells.ConclusionsThe data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

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High-Level Expression, Purification, and Renaturation of Recombinant Murine Interleukin-2 from Escherichia coli

Cited by 18 publications

References 0 publications

Rainbow trout interleukin-2: Cloning, expression and bioactivity analysis

Rainbow trout interleukin-2: Cloning, expression and bioactivity analysis

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells

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