Fluorescence-Activated Cell Sorting of Human L-asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity
Fluorescence-Activated Cell Sorting of Human L-asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity
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DOI：
10.1021/acschembio.6b00283
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发表时间：
2016-09-01
影响因子：
4
通讯作者：
Konrad, Manfred
中科院分区：
文献类型：
--
作者：
Karamitros, Christos S.;Konrad, Manfred
免疫原性是使用非人源蛋白质药物治疗期间最常见的并发症之一。这种情况的一个值得注意的例子是用于治疗急性淋巴细胞白血病（ALL）的细菌L-天冬酰胺酶（L-ASNases）。用人类酶代替细菌酶被认为是抗白血病治疗的重大改进的基础。最近，我们解决了具有L-ASNase活性的人酶的晶体结构，命名为hASNase-3。这种酶表达为无活性的前体蛋白，并在后处理过程中经历分子内加工，导致产生两个亚基，这两个亚基保持非共价但紧密结合，并构成酶的催化活性形式。我们发现，这种分子内加工可以通过游离氨基酸甘氨酸显著地和选择性地加速。在本研究中，我们报告的hASNase-3的分子工程，旨在提高其催化性能。我们创建了一个基于荧光激活细胞分选（FACS）的高通量筛选系统，用于表征合理设计的突变体文库，利用游离甘氨酸促进自蛋白水解切割的发现，其激活在大肠杆菌中表达的突变蛋白。缺乏天冬氨酸生物合成的大肠杆菌菌株。连续的筛选轮次导致分离出催化改进的变体，其显示出与野生型酶相比高达6倍的更好的催化效率。我们的工作建立了一个强有力的战略，进一步利用人类天冬酰胺酶序列空间，以促进体外进化的酶物种的鉴定，这将奠定基础，改善ALL治疗。
Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial L-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.