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Fig. 1 | BMC Biotechnology

Fig. 1

From: Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins

Fig. 1

Comparison of previously reported SPI fusion protein cloning protocol with the new protocol outlined in this work. (A) Previously reported cloning protocol to construct a SPI fusion protein from pET-SUMO and pTXB1 vectors (Lamer et al., 2022a). The desired peptide gene is amplified with PCR and inserted into the linearized pET-SUMO vector with TA cloning. The His6-SUMO-peptide gene is then amplified with PCR, and flanking 5’ NdeI and 3’ SapI restriction sites are inserted with appropriate DNA primer design. The PCR product and pTXB1 plasmid are both doubly digested with NdeI and SapI, and then ligated together to construct the pTXB1-SPI plasmid with the target peptide gene inserted seamlessly between His6-SUMO and intein-CBD genes. (B) Vector map for pSPIH6, based on the pTXB1 backbone. The His6-SUMO and intein-CBD-His6 genes are separated by a PacI restriction site to allow insertion of a target peptide gene. An ampicillin resistance gene acts as a selective marker. Expression of a SPI fusion protein is under the control of a T7 promoter, and can be induced with the addition of IPTG to the culture media. (C) Simplified cloning protocol used in this work to construct a SPI fusion protein from pSPIH6. The plasmid is first digested with PacI. The target peptide gene is amplified with PCR, and DNA primers are designed to create 15–30 bp overlaps on the 5’ and 3’ ends of the target gene sequence. These 5’ and 3’ overlapping regions are identical to the 3’ end of the His6-SUMO gene and the 5’ end of the intein-CBD-His6 genes, respectively, to allow for NEBuilder® HiFi DNA Assembly with the linearized pSPIH6 vector. The bases of the PacI restriction site are removed with 5’ and 3’ exonuclease activity of the DNA Polymerase included in the NEBuilder® kit to create a seamless SPI gene in the newly constructed pSPIH6-peptide plasmid

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