High efficient expression and purification system of small protein structure field in colibacillus
A technology of expression vectors and elements, which is applied in the field of protein expression and purification systems, and can solve the problems of poor solubility and easy degradation of small peptides
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Embodiment 1
[0078] Example 1 Construction of expression vectors pGBTNH and pGBH
[0079] GB1 fusion expression vectors pGBTNH and pGBH, with GB1 gene (A.M.Gronenborn et al., A novel, highly stable fold of the immunoglobulin binding domain of streptococcal proteinG, Science 253 (1991) 657-661) as template, based on pET-22b vector (Novagen ) constructed from. The structure of the pGBTNH vector is shown in Figure 1, in the induced T 7 After the RNA polymerase promoter (omitted in the figure), it sequentially includes a GB1 tag, a linker, a thrombin cleavage site, a multiple cloning site and six histidine tags at the carboxy-terminal. The structure of pGBH is the same as that of pGBTNH except that there is no linker and thrombin cleavage site. And, pGBTNH, encodes the amino acid sequence including the SSGLVPR fragment before the asterisk (Fig. 1). PGBH, encoding the amino acid sequence excluding the asterisk.
[0080] When constructing the pGBTNH plasmid, the DNA fragment encoding the thr...
Embodiment 2
[0091] The preparation of embodiment 2 small domains or fragments
[0092] In this example, eight small domains or fragments were selected for testing. The eight cDNA fragments encoding each small domain or fragment were amplified by PCR, and the sources of each small domain or fragment were as follows:
[0093] CUE domain (CUEa, CUEb) from human Tollip protein;
[0094] WW1 and WW2 domains are derived from human FBP-11 protein;
[0095] UIM is derived from human S5a protein;
[0096] DATc is derived from the carboxy-terminal cytoplasmic tail of the murine dopamine transporter;
[0097] α-Syn 31-109 and α-Syn 61-95 α-Syn (alpha-synuclein) from human.
[0098] The above PCR product was cloned into the BamH I and Xho I cloning sites of the bacterial expression vector pGBTNH or pGBH prepared as in Example 1 by conventional methods, see Table 1.
[0099] Table 1 Structural peptide regions and related information used in the present invention
[0100] Peptide name ...
Embodiment 3
[0102] Example 3 Expression and purification of GB1 fusion protein
[0103] The expression vector containing the structural peptide gene obtained in Example 2 was transformed into Escherichia coli BL21 (DE3) competent cells by conventional transformation methods.
[0104] For the expression and purification of the fusion protein, see the following steps:
[0105] Transfer 10 mL of overnight bacteria to 1 L of LB medium and shake at 37°C. When the OD of the bacteria 600At about 0.7, 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) was added to induce protein expression, and placed on a shaker at 22° C. for 12 hours. The bacteria were collected by centrifugation (5000rpm, 10 minutes, 4°C), resuspended with buffer A (50mM phosphoric acid, 300mM NaCl, pH8.0, 0.5mM PMSF (phenylmethylsulfonyl fluoride)), ultrasonically lysed and centrifuged ( 15,000 rpm, 15 minutes, 4°C). Lysates, supernatants and pellets were analyzed by SDS-PAGE (15% gel) and stained with Coomassie. The supernatan...
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