The sequence of CaSPDS was obtained from the cloning vector by using EcoRI and SaI1 restriction endonucleases (TransGen Biotech, Beijing, China) and then connected into pCold I vector (TaKaRa, Tokyo, Japan) by T4 ligase (TransGen Biotech, Beijing, China), producing pCold-CaSPDS. The pCold I empty vector and validated pCold-CaSPDS were transfected into the Escherichia coli BL21 (DE3) cells (TaKaRa, Dalian, China) and then cultured in Luria–Bertani (50 µg/mL Amp) liquid medium at 37 °C with shaking at 200 rpm until the OD600 reached 0.6. Different concentrations of isopropyl-β-d-thiogalactoside (IPTG, 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM) were added for 24 h to induce expression. It was then induced at the optimum IPTG concentration for 0, 2, 6, 12, 24, and 36 h. The ultrasonic fragmentation and purification of the CaSPDS recombinant protein were conducted as previously reported [48 (link)].
Escherichia coli bl21 de3 cells
Manufactured by Takara Bio
Sourced in China, Japan
Escherichia coli BL21 (DE3) cells are a widely used bacterial strain commonly employed in recombinant protein expression. They are designed for high-level expression of target proteins under the control of the T7 promoter system.
Automatically generated - may contain errors
2 protocols using escherichia coli bl21 de3 cells
1
Cloning and Expression of CaSPDS
2
Cloning and Sequencing TSHSV-661 Gene
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The purified TSHSV-661 segment was digested using BamHI and XhoII restriction enzymes at 37 ℃for 3 h and the digested products were inserted into the pET-30a vector (TaKaRa, China) via a ligase enzyme, and the recombinant plasmid was named as pET30a-TSHSV-661. The recombinant plasmid was transformed directly into Escherichia coli BL21 (DE3) cells (TaKaRa, Japan). The successful construction of the pET30a-TSHSV-661 clone was confirmed by PCR amplification using the above specific primers by the same PCR reaction, and amplified products were checked via agarose gel electrophoresis. The plasmid of the positive pET30a-TSHSV-661 clone was extracted for sequencing (Sunya, China).
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