Method for preparing a target protein using the sHSPs
一种目的、蛋白的技术，应用在生物转化领域，能够解决尚未公布降解等问题
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Embodiment 1
[0038] Embodiment 1: the preparation of the recombinant plasmid that comprises ibpA, ibpB or HSP26 gene
[0039] E.coli W3110 (ATCC 39936), Pseudomonas putida (Pseudomonas putida) KT2440 (ATCC 47054) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) chromosomal DNA according to the method of Sambrook et al. (Molecular cloning, second edition, Cold Spring Harbor Laboratory Publishing House (ColdSpring Harbor Laboratory Press), NY, 1989) for isolation and purification.
[0040] E. coli W3110, Pseudomonas putida (Pseudomonasputida) KT2440 and Saccharomyces cerevisiae were respectively cultured in 500 ml of LB (Luria-Bertani) medium for 24 hours. The strains in the early exponential growth phase were collected by centrifugation, and then suspended in 50 ml of TE solution (10 mM Tris, 1 mM EDTA; pH 7.6) containing 10 mg / mL lysozyme. The strain suspension was cultured with slow shaking at room temperature for 24 hours.
[0041] In order to destroy the strain and remove the ...
Embodiment 2
[0060] Embodiment 2: Purification of IbpA, IbpB and HSP26 protein
[0061] Transformed with recombinant plasmids pTac99IbpAH, pTac99IbpBH, pTac99 PP E.coli XL 1-Blue (Stratagene, USA) of IbpAH and pTac99HSP26H were carried out in LB medium containing 50mg / L ampicillin (yeast extract 5g / L, tryptophan 10 / L, NaCl 10g / L) Cultivation, the above-mentioned recombinant plasmids respectively include the coding genes of IbpA, IbpB or HSP26 protein in Example 1.
[0062] When the optical density (O.D.) of the spectrophotometric detection at 600nm reaches 0.7, 1mM IPTG (isopropyl-β-thiogalactoside) is added to induce the expression of IbpA, IbpB, PP IbpA and HSP26 proteins. After 4 hours of induction, 1 mL of each medium was taken and centrifuged at 6000 rpm at 4°C for 5 minutes, and then the obtained precipitate was washed with 0.5 mL of TE solution (10 mM Tris-HCl, 1 mM EDTA; pH 8.0) and Centrifuge at 6000 rpm, 4°C for 5 minutes to obtain a pellet. The precipitate was suspended in...
Embodiment 3
[0066] Example 3: The effect of sHSPs on the degradation process of the target protein caused by trypsin
[0067] Since the target protein is easily attacked by proteases in the cell lysate, the target protein is severely lost. In this example, human serum albumin as the protein of interest was diluted in a cell lysate, followed by incubation for 2 hours at room temperature in a solution containing various concentrations of trypsin as a protease. The concentration of protease relative to the protein of interest was changed to 0, 1 / 10, 1 / 20, 1 / 30, and 1 / 50. IbpA and IbpB derived from E. coli and HSP26 derived from Saccharomyces cerevisiae were used as sHSPs.
[0068] Fig. 6 is an electrophoresis graph showing the inhibitory effect of sHSPs on protease in the cell lysate supplemented with human serum albumin. As shown in Figure 6, the target protein, human serum albumin, was hardly degraded in the solution containing sHSPs, while on the other hand, most of the human serum al...
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