Construction of new ligation-independent cloning vectors for the expression and purification of recombinant proteins in silkworms using BmNPV bacmid system.
Construction of new ligation-independent cloning vectors for the expression and purification of recombinant proteins in silkworms using BmNPV bacmid system.
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DOI：
10.1371/journal.pone.0064007
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Park EY
中科院分区：
文献类型：
--
作者：
Kato T;Thompson JR;Park EY
为了在家蚕表达系统中快速、高效地克隆基因，建立了一种非连接依赖性克隆系统（LIC）。通过在家蚕幼虫和蛹中表达人微粒体甘油三酯转移蛋白（hMTP）和EGFP，证实了该系统的有效性。此外，将hMTP和人蛋白质二硫键异构酶（hPDI）基因插入到两个含有gcLINK序列的LIC载体中，并通过使用LIC通过gcLINK序列进行组合。将构建的载体整合到家蚕核型多角体病毒（BmNPV）杆粒中，并注射到家蚕幼虫中。从家蚕幼虫脂肪体中纯化表达的hMTP-hPDI复合物。利用该LIC载体系统在家蚕幼虫中表达人α-酮戊二酸脱氢酶（KGDH）E1、E2和E3亚基。使用三种不同的亲和层析步骤从脂肪体中容易地纯化表达的蛋白质。如本报告所述构建的LIC载体允许通过使用BmNPV杆粒系统快速表达和纯化重组蛋白或其复合物。
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.
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