Abstract
A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two LIC vectors harboring gcLINK sequences and were combined by using the LIC through gcLINK sequences. The constructed vector was incorporated into the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, and injected into silkworm larvae. The expressed hMTP-hPDI complex was purified from the fat bodies of silkworm larvae. This LIC vector system was applied to express the E1, E2, and E3 subunits of human α-ketoglutarate dehydrogenase (KGDH) in silkworm larvae. The expressed proteins were purified easily from fat bodies using three different affinity chromatography steps. The LIC vectors constructed as described in this report allow for the rapid expression and purification of recombinant proteins or their complexes by using the BmNPV bacmid system.
Highlights
To produce recombinant proteins, target genes are amplified by PCR with primers containing restriction enzyme sites
The human microsomal triglyceride transfer protein (hMTP)-hPDI complex was expressed in silkworm larvae using newly constructed Ligation-independent cloning (LIC) vectors. hMTP is located in the lumen of endoplasmic reticulum (ER) as a heterodimer with human protein disulfide isomerase [16,17]
The hMTP gene could be inserted with high efficiency at the LIC site by using the LIC reaction
Summary
To produce recombinant proteins, target genes are amplified by PCR with primers containing restriction enzyme sites. PCR products and expression vectors are digested by restriction enzymes and ligated using T4 DNA ligase. Using this protocol, the available restriction enzymes depend on the nucleotide sequences of target genes, and different enzymes should be used when many target genes are cloned in parallel. Ligation-independent cloning (LIC) has been developed as a new cloning method, which eliminates the use of restriction endonuclease digestion and ligation of PCR products [1,2]. LIC circumvents the limitations of traditional gene cloning methods because any PCR products can be inserted into LIC-compatible cloning vectors without restriction enzymes and T4 DNA ligase. PCR products and vectors are treated with T4 DNA polymerase in the presence of a single deoxyribonucleotide triphosphate, which generates specific 12–20 nucleotide single stranded overhangs.
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