A small molecule protein that efficiently mediates the formation of inclusion bodies from recombinant polypeptides
A technology of small molecular proteins and recombinant polypeptides, which is applied in the field of bioengineering, can solve problems such as cumbersome operations, and achieve the effects of simplifying the separation and purification process, remarkable effects, large market value and application prospects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0045] Example 1 Construction of pET28b-Am-Metch expression vector
[0046] (1) In vitro synthesis of a gene encoding a small molecule protein (Am) that efficiently mediates the formation of inclusion bodies from recombinant polypeptides
[0047] According to the hydrophobic and hydrophilic properties of different amino acids and their tendency to form β-sheets, the C-terminal sequence (amino acids 100-161) (PagP-100) of Escherichia coli palmitoyl phospholipid transferase (PagP) was optimized and modified. Fusion tags (Am) of inclusion bodies were formed, and the nucleotide sequence of the gene encoding Am was designed according to the codon bias of E. coli. Among them, the nucleotide sequence of the gene encoding Am was synthesized by Beijing Ruibo Biotechnology Co., Ltd. Guangzhou Branch.
[0048] Nucleotide sequence of PagP-100:
[0049] AATTTTCATTTAGGTCTGGGATTCACCGCTGGCGTAACGGCACGCGATAACTGGAATTACATCCCTCTCCCGGTTCTACTGCCATTGGCCTCCGTGGGTTATGGCCCAGTGACTTTTCAGATGACCTACATTCCGG...
Embodiment 2
[0075] Example 2 Induction of recombinant polypeptide expression in Escherichia coli in the form of inclusion bodies
[0076] The pET28b-Am-Metch expression vector constructed above and the control pET28b-PagP-100-Metch expression vector (PagP-100 instead of Am) were transformed into E. coli BL21 (DE3) competent cells, and a single clone was picked and inoculated into liquid LB Incubate overnight in medium and expand to OD by volume ratio of 1:100 600 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.6 mM, and induce protein expression at 37°C for 24h. The cells were collected by centrifugation at 5000 g, 10 min, and 4°C to obtain cells expressing recombinant polypeptides.
[0077] The expression of recombinant polypeptide was detected by 18% SDS-PAGE, and the results were as follows figure 2 As shown, the expression level of Am-Metch was significantly better than that of PagP-100-Metch.
[0078] 18% SDS-PAGE was used to detect the exp...
Embodiment 3
[0080] Example 3 Dissolution of inclusion bodies and separation and purification of target polypeptide Metch
[0081] (1) with 1 × Lysis Buffer (50 mmol NaH 2 PO 4 , 300mmol NaCl, 20mmol imidazole, pH8.0) suspended the thalline expressing the recombinant polypeptide collected in Example 2, sonicated it, at 5000g, 30min, 4°C, centrifuged to get the precipitate to obtain the inclusion body expressing the recombinant polypeptide ;
[0082] (2) Mix the inclusion body with 100 or 200 mmol acetic acid solution at the ratio of 10 mg: 1 ml to realize the dissolution of the inclusion body. The solubilization of inclusion bodies in solubilized samples was detected by SDS-PAGE, such as Figure 5 As shown, the dissolution effect of Am-Metch inclusion bodies was good after the dissolution of 200 mmol acetic acid solution.
[0083] (3) Add Tween-20 at 2% volume percentage to the dissolved sample, shake at 37°C for 2 hours to further promote dissolution; then use NaOH to adjust the pH to...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More