Neuhold Jana, Radakovics Katharina, Lehner Anita, Weissmann Florian, Garcia Maria Queralt, Romero Mari Carmen, Berrow Nicholas S, Stolt-Bergner Peggy
Protein Technologies Facility, Vienna BioCenter Core Facilities, Dr. Bohr-gasse 3, 1030, Vienna, Austria.
Research Institute of Molecular Pathology, Campus Vienna Biocenter 1, 1030, Vienna, Austria.
BMC Biotechnol. 2020 May 12;20(1):26. doi: 10.1186/s12896-020-00616-z.
Recombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as "GoldenBac".
This method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2-15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein.
This robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.
在真核生物中生产和纯化大型蛋白质复合物的重组蛋白需要高效的方法来生成多基因表达构建体，其中每个单独的基因都由其自身的启动子和终止子控制。目前的方法要么基于通过Cre-lox技术对含有loxP位点的多个载体进行连续轮次的组合，要么基于通过PCR或其他方法进行多轮基因组合。这些方法是多步骤的，效率低于单基因克隆，并且可能需要费力的过程来验证最终产物中是否存在所有感兴趣的基因。在此，我们描述了一种基于金门的快速简单系统，用于使用Tn7转座或基于KO1629的同源重组生成与杆状病毒表达载体系统（BEVS）兼容的多基因表达构建体，我们将其称为“GoldenBac”。
该方法基于构建一系列载体，这些载体包含一个由BsaI IIS型限制酶切割位点侧翼的启动子-感兴趣基因-终止子盒。这一系列载体可以被BsaI切割以切除具有独特突出端的盒。在同一反应中，然后将这些盒以正确的顺序连接到最终目的载体中，以生成包含2至15个基因的多基因表达构建体。因此，单个表达构建体可以在单个反应中组合到单个载体中，当组合多达14个表达盒时效率超过90%。我们展示了三种不同共表达系统的成功构建和表达，即蛋白酶体盖子复合物、后期促进复合物/细胞周期体（APC/C），以及一系列用于测试伴侣共表达对HOIP蛋白溶解度影响的构建体。
这种强大的单步克隆系统为基于转座和同源重组的杆状病毒系统生成多基因表达构建体提供了一种易于使用的方法，使该技术可供所有使用杆状病毒表达系统的实验室使用。这种高效且简单的方法允许将多基因表达克隆快速纳入蛋白质生产设施的标准化服务组合中，并且任何实验室也可以轻松采用该方法用于常规生成多基因杆状病毒构建体。
