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Pet28a expression vector

Manufactured by Qiagen
Sourced in Germany

The pET-28a expression vector is a plasmid used for the expression of recombinant proteins in Escherichia coli. It contains a T7 promoter, a lacI gene, and a kanamycin resistance marker. The vector can be used to produce N-terminal or C-terminal His-tagged fusion proteins.

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3 protocols using pet28a expression vector

1

Cloning and Expression of FliD Protein

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EXAMPLE 1

All DNA manipulations were performed under standard conditions as described by Sambrook et al. (Sambrook, et al., 1989). Briefly, the FliD gene was amplified by PCR using genomic DNA from H. pylori strain J99 as the template. Following oligonucleotides were used as primers: 5′-CAT ATG GCA ATA GGT TCA TTA A-3′ (SEQ ID NO: 19) and 5′-CTC GAG ATT CTT TTT AGC CGC TGC-3′ (SEQ ID NO: 20). Using this approach a NdeI site was introduced at the 5′-end of forward primers and a XhoI site at 5′-end of the reverse primers. After PCR amplification, the product (2058 bp) was ligated into the pTZ57R/T cloning vector (InsTAclone™ PCR Cloning Kit, MBI Fermentas, Lithuania). Subsequently, the insert was confirmed via PCR and sequencing, and was cloned into a PET-28a(+) expression vector (Qiagen, USA) using NdeI and XhoI restriction enzymes.

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2

Recombinant Protein Expression Protocol

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pGEM-T Easy vector, DNA polymerase and DNA extraction kit were obtained from Promega (Madison, WI, USA). Plasmid isolation kit, pET28a expression vector, and nickel-nitrilotriacetic acid (NiNTA) superflow column were purchased from Qiagen (Hilden, Germany). The restriction enzymes (BamHI and SalI) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, USA). And all other chemicals for assays were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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3

Molecular Cloning and Protein Purification

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Ex-Taq DNA polymerase, a genomic DNA extraction kit, pGEM-T easy vector, and reagents for polymerase chain reaction were purchased from Promega (Madison, WI, USA). T4 DNA ligase and restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The pET28a expression vector, plasmid isolation kit, and nickel-nitrilotriacetic acid superflow column for His-tag protein purification were obtained from Qiagen (Hilden, Germany)20 (link). Oligonucleotide primers were obtained from Bioneer (Daejeon, Republic of Korea)29 (link). Electrophoresis reagents were provided by Bio-Rad (Hercules, CA, USA), and all chemicals used in assays were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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