CCHFV Kelkit06 NP (1449 bp) retrieved from GenBank (Accession no. GQ337053) was optimized for bacterial expression, synthesized, and cloned into pUC19 by GenScript USA Inc. (Nanjing, Jiangning, China) and then, propagated in DH5α Escherichia coli cells. For the expression, CCHFV NP was subcloned into pET28b (Addgene, Cambridge, USA) by using XhoI (Sigma-Aldrich, Taufkirchen, Germany) and NcoI (New England Biolabs, Massachusetts, USA) restriction enzymes and transformed into One Shot BL21(DE3) Chemically Competent E. coli cells (Thermo Fisher Scientific, Waltham, USA). After transformation, positive transformants were selected with kanamycin and analyzed by sequencing.
Xhoi
Manufactured by Merck Group
Sourced in United States
XhoI is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-CTCGAG-3'. It is commonly used in molecular biology and genetic engineering applications for the digestion and manipulation of DNA.
Automatically generated - may contain errors
3 protocols using xhoi
1
Bacterial Expression of CCHFV Nucleoprotein
2
Plasmid Isolation and Verification
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A 5446 bp plasmid pc-DNA3-EGFP (Addgene plasmid # 13031—a gift from Doug Golenbock), encoding for EGFP was produced in Escherichia coli cells, amplified in lysogeny broth, following the protocol provided by Addgene. The plasmid was extracted and purified using a Qiagen purification kit (Cat. #12381), as per manufacturer’s instructions. The purity of the pDNA preparation was assessed using a Nanodrop spectrophotometer via 230/260 nm and 260/280 nm measurements. To confirm that the right plasmid was obtained, a restriction enzyme digest was performed. The pDNA was digested with two restriction enzymes, XbaI and XhoI (Sigma Aldrich), run on a 4% (w/v) agarose gel containing 2 µL SYBR® Safe DNA Gel Stain, and electrophoresed at 100 V for 90 min in a Midi plus-1 Horizontal Electrophoresis System, (ME10-7-10, Major Science, Saratoga, CA, USA) coupled with BioRad Power Pac 200 (Bio Rad, CA, USA) electrophoresis power supply.
3
Optimized Expression of Recombinant Protein
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The amino acid sequence of rSNTX was subjected to reverse translation into a nucleic acid sequence using EMBOSS Backtranseq (https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/), with codon usage tailored for E. coli K12 -a widely recognized host organism for recombinant protein expression. To further optimize the genetic construct for robust and e cient protein expression, meticulous gene optimization parameters were subsequently applied. Both the optimized and unmodi ed versions of the rSNTX sequences were scrutinized using the specialized GenScript Rare Codon Analysis Tool (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis). Additionally, the levels of free energy, secondary structure thermodynamics, and mRNA folding were compared before and after gene optimization using The UNAFold Web Server (http://www.unafold.org/mfold/applications/rna-folding-form.php). This analysis demonstrates that the optimization does not signi cantly alter the mRNA structure in a way that affects protein expression. Finally, the trxA and 6x His-tag sequences were added to the N-terminal and C-terminal of the optimized sequence, respectively. SnapGene software was utilized to precisely determine the insertion site for rSNTX, and we selected the EcoRI and XhoI restriction enzymes (Sigma, USA) for subsequent cloning steps.
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