Method for producing polypeptide by combining biological expression of protein with chemical cracking technique
A technology for chemical cleavage and protein binding, applied in the field of polypeptide preparation, can solve the problems such as the inability of the technology to be widely used, the inability to cleave the inclusion body protein, and the difficulty of purification, so as to achieve easy industrialization implementation, large industrialization prospects, and low cost. Effect
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Embodiment 1
[0027] Example 1 Construction of expression vector
[0028] Gene synthesis of Staphlococcal nuclease-polypeptide (Staphlococcal nuclease-Peptide) base sequence (sequence shown in SEQ ID NO:1, commissioned by Shanghai Jierui Bioengineering Co., Ltd.); gene synthesis of Staphlococcal nuclease-Peptide The base sequence method is based on conventional gene synthesis methods, such as synthesizing a set of primers based on the base sequence of Staphlococcal nuclease-Peptide, and then performing PCR amplification respectively, and finally splicing the entire target gene. After that, the base sequence (SEQ ID NO:1) and pET21a expression vector sequence (purchased from Novagen) were synthesized by double digestion with restriction endonuclease sites Nde I and XhoI, and the DNA quick ligation kit (GK6001) (Purchased from Shanghai Jierui Biological Engineering Co., Ltd.) Connect the two digested sequences, and this is the constructed expression vector. The constructed expression vector was...
Embodiment 2
[0029] Example 2 Transform the expression vector into an expression host, and induce the expression of recombinant polypeptide
[0030] The expression vector was transformed into the expression host Escherichia coli BL21(DE3) (purchased from Shanghai Jierui Bioengineering Co., Ltd.) by the heat shock method (reference molecular cloning) to obtain the recombinant genetic engineering expression strain BL21(DE3) / SNase-peptide-pET21a . Cultivate the expression strain BL21(DE3) / SNase-peptide-pET21a at 37°C to an OD700nm of approximately 1.0, and add IPTG (isopropyl-β-D-thiogalactoside, purchased from Shanghai Shenggong Biotechnology Co., Ltd. Engineering Technology Service Co., Ltd.), induce expression at 37°C for 4 hours, see the results of induced expression figure 1 .
Embodiment 3
[0031] Example 3 Collection and disruption of bacteria, purification of expressed recombinant polypeptide
[0032] The induced expression strain BL21(DE3) / SNase-peptide-pET21a was collected by centrifugation, the bacteria was disrupted by ultrasound, and centrifuged at 13000 rpm for 20 minutes. The collected precipitate was resuspended in 50mM tirs, 8MUrea, pH8.0 buffer, stirred at room temperature for 1 hour, and centrifuged at 13000rpm for 20 minutes. The supernatant was passed through SP-sepharose (SP-Sepharose) column. Specific steps are as follows:
[0033] 1, 3-5 times the volume of 50mM tirs, 8M Urea, pH8.0 buffer balance SP-sepharose;
[0034] 2. Load the supernatant on the column;
[0035] 3. Wash SP-sepharose with 3-5 times volume of 50mM tirs, 8M Urea, pH8.0 buffer;
[0036] 4. Wash SP-sepharose with 0.05mM NaCl, 50mM tirs, 8M Urea, pH8.0 buffer;
[0037] 5, 0.08mM NaCl, 50mM tirs, 8M Urea, pH8.0 buffer to elute Staphlococcal nuclease-Peptid (Staphlococcal nuclease-Peptid) ...
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