aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)

Detailní popis

The Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.

The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.

Highlights

• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile – tagged or untaged protein expression with tag removal option

Applications

• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes

Principle

The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.

The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts (see Reference 2). T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand (see Figure 2). After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.

Features

The system consists of four kits based on the pLATE series of bacterial expression vectors:

• aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
• aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK) – pLATE51 vector, N-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag) – pLATE31 vector, C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ) – pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
• aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK) – pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
• aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ) – pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.

For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.

Notes

pLATE bacterial expression vector sequences

• pLATE11 sequence in TXT format (pLATE11_seq.txt) or GenBank format (pLATE11-ready-to-use.gb)
• pLATE31 sequence in TXT format (pLATE31_seq.txt) or GenBank format (pLATE31-ready-to-use.gb)
• pLATE51 sequence in TXT format (pLATE51_seq.txt) or GenBank format (pLATE51-ready-to-use.gb)
• pLATE52 sequence in TXT format (pLATE52_seq.txt) or GenBank format (pLATE51-ready-to-use.gb)

Control PCR fragments

• for pLATE11 in TXT format or GenBank format
• for pLATE31 in TXT format or GenBank format
• for pLATE51 in TXT format or GenBank format
• for pLATE52 in TXT format or GenBank format