Method for solubilizing insoluble proteins and/or peptides
A protein and soluble technology, which is used in the reagents for inducing cytotoxic T lymphocytes, preventing or treating cancer and/or infectious diseases, and can solve problems such as inability to obtain effects, protein precipitation, and protein insolubility
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Embodiment 1
[0148] Nucleotide Availability Studies
[0149] Recombinant mouse tyrosinase-related protein 2 (hereinafter, also referred to as "TRP-2") was used as an insoluble protein, and a solubilization method using nucleotides was investigated.
[0150] First, a recombinant fusion protein composed of a ubiquitin fragment (hereinafter referred to as "Ubi") and TRP-2 (hereinafter referred to as "Ubi") with a histidine tag (His-tag) added at the N-terminus was prepared UT"). Ubi was isolated from cDNA encoding the amino acid sequence described in SEQ ID NO: 1, and TRP-2 was isolated from the N-terminal 56th residue glutamic acid encoding based on the amino acid sequence described in ACNo.P29812 of the SWISS-PROT database (E) cDNA from serine (S) to the 472nd residue at the N-terminus. Ubi and TRP-2 were inserted into pET19b vector (Novagen) to form an expression plasmid (pET19b / UT). When Escherichia coli (Rosetta-gami2(DE3)pLysS: Novagen, #71352-3) was transformed with pET19b / UT to exp...
Embodiment 2
[0182] Nucleotide species (base) research
[0183] As a model of insoluble protein, recombinant mouse TRP-2 (rTRP-2; SEQ ID NO: 3) was used. rTRP-2 was isolated from a protein encoding a glutamic acid (E) at the 56th residue at the N-terminus to a serine (S) at the 472nd residue at the N-terminus based on the amino acid sequence described in ACNo.P29812 of the SWISS-PROT database cDNA, and inserted into the pET19b vector (Novagen) to form an expression plasmid (pET19b / mdT). When Escherichia coli (Rosetta-gami2(DE3)pLysS: Novagen, #71352-3) was transformed with pET19b / mdT and the recombinant was expressed, a recombinant protein having a histidine tag (His-tag) at the N-terminal was obtained. The recombinant protein forms inclusion bodies and is insoluble. The insoluble fraction was dissolved in 8M urea / PBS / 5mMDTT (pH8.5) or 8M urea / PBS / 10mMDTT (pH8.0), and passed through the nickel affinity (HisTrapHP column) using AKTAexplorer liquid chromatography system (AmershamPharmasiaB...
Embodiment 3
[0187] Research on the types of nucleotides (ribonucleotides and deoxyribonucleotides)
[0188] rTRP-2 dissolved in 6M urea / PBS / 5mMDTT (pH 8.5) was prepared in 6 sample tubes and 2.5mMGSH and 2.5mMGSSG were added to all tubes. GTP (Sigma, #G8877) or dGTP was added to each tube at a concentration of 2 mM, 10 mM or 50 mM and all tubes were maintained for 1 hour. Afterwards, 10% glycerol was added to each tube. Dialysis was performed against 10% glycerol / PBS (pH 8.0), and the solvent was replaced. Thereafter, the solution was centrifuged at 15100 x g for 1 hour at 10° C. and fractionated into a supernatant and a precipitate, and then analyzed similarly to Example 1 by SDS-PAGE.
[0189] as in image 3 As shown in , at 2 to 10 mM GTP and 2 to 10 mM dGTP, about 50% was dissolved and recovered in the supernatant. However, when the concentration of GTP and dGTP reached 50 mM, almost all amounts were insoluble and recovered in the precipitated fraction. From the above, it is cons...
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