The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts’ metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host’s viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors’ background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3′-protruding). The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

中文翻译：

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促进有毒蛋白质的生物合成：工程细菌碱性磷酸酶的体内瞬时失活。

酶的生物技术生产经常因克隆和表达的基因的重组产物的毒性而困扰，这会干扰重组宿主的代谢。已经采取了各种方法来克服这些限制，例如严格控制重组基因或分泌重组蛋白。工业生产蛋白质的方法要求最大可能的生物合成蛋白质产量，并与重组宿主的生存能力保持平衡。大肠杆菌（E. coli）的细菌碱性磷酸酶（BAP）是用于蛋白质/抗体检测和分子克隆的关键酶。由于可从DNA，RNA和脱氧核糖核苷三磷酸中去除末端磷酸酯，因此可用于降低自连接载体的背景。前体酶在N端包含一个信号肽，并分泌到大肠杆菌周质中。然后，将前导物剪下并在氧化时形成二聚体。我们提出了一种新的方法来对phoA基因进行克隆，工程化，表达，纯化和瞬时失活酶的再激活。通过用N端His6-tag替换分泌前导区编码部分来修饰重组bap基因，在PBAD启动子表达载体中克隆并在大肠杆菌中表达。基因表达稳定，导致His6-BAP在细胞质中积累，超过细胞总蛋白的50％。His6-BAP蛋白对细胞无害，因为其天然毒性被大肠杆菌细胞质内的还原性环境抑制，阻止了活性酶的形成。一个基于沉淀和固定金属亲和色谱（IMAC）纯化的简单方案产生了均质蛋白，该蛋白通过透析被还原到氧化还原缓冲液中，该氧化还原缓冲液包含还原和氧化的巯基化合物以及稳定蛋白质结构的辅因子Zn2 +，Mg2 +和磷酸盐。重组的His6-BAP表现出高活性，被用于开发针对所有类型的DNA末端的有效方案，包括有问题的末端（钝的，3'突出的）。所开发的方法似乎非常适合于超纯BAP的工业生产。此外，通过在细胞质中以无活性的状态进行生物毒性合成而使分泌的毒性酶瞬时失活的方法，通常可以应用于其他有问题的蛋白质。