Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

Benevides C. C. Pessela; Alejandro Vian; César Mateo; Roberto Fernández-Lafuente; José L. García; José M. Guisán; Alfonso V. Carrascosa

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Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

机译：Thermus sp。的生产过剩。大肠杆菌中的菌株T2β-半乳糖苷酶及其定制金属螯合物载体的制备

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A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.

机译：通过将聚His标签融合到Thermus sp。的β-半乳糖苷酶的N端区域，构建了一种新型的热稳定嵌合β-半乳糖苷酶。 T2菌株T2，以促进其在大肠杆菌中的过表达以及通过固定的金属离子亲和色谱（IMAC）进行纯化。聚组氨酸标签融合不影响β-半乳糖苷酶的活化，动力学参数和稳定性。亚氨基二乙酸铜（Cu-IDA）载体可使His-tagged酶最快速地吸附，促进多亚基相互作用，但对酶的稳定性造成不利影响。为了改善酶的纯化，通过设计定制的低活化Co-IDA或Ni-IDA载体可以实现选择性的单点吸附。这种新酶不仅可用于工业用途，而且已成为研究通过选择性吸附在特制IMAC载体上纯化大型多聚体蛋白质的极佳模型。

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《Applied and Environmental Microbiology》 |2003年第4期|共6页
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Benevides C. C. Pessela; Alejandro Vian; César Mateo; Roberto Fernández-Lafuente; José L. García; José M. Guisán; Alfonso V. Carrascosa;
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1. Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli [J] . Mitsunori Ishiguro, Satoshi Kaneko, Atsushi Kuno, Applied and Environmental Microbiology . 2001,第4期

机译：重组Thermus sp。的纯化和鉴定。在大肠杆菌中表达的菌株T2α-半乳糖苷酶
2. Engineering the D-amino acid oxidase from Trigonopsis variabilis tofacilitate its overproduction in Escherichia coli and its downstreamprocessing by tailor-made metal chelate supports [J] . Alonso J., Barredo JL., Armisen P., Enzyme and Microbial Technology . 1999,第1a2期

机译：工程化Trigonopsis variabilis的D-氨基酸氧化酶，以促进其在大肠杆菌中的过量生产以及通过量身定制的金属螯合载体进行下游加工
3. Engineering the D-amino acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its downstream processing by tailor-made metal chelate supports [J] . Alonso Jorge, Armisen Pilar, Diez Bruno, Enzyme and Microbial Technology . 1999,第1期

机译：工程化Trigonopsis variabilis的D-氨基酸氧化酶，以促进其在大肠杆菌中的过量生产以及通过量身定制的金属螯合载体进行下游加工
4. APOOBELIN BIOTINYLATED IN VIVO: OVERPRODUCTION IN ESCHERICHIA COLI CELLS [C] . SV MARKOVA, GA STEPANYUK, LA FRANK, 12th Symposium on Bioluminescence and Chemiluminescence, Apr 5-9, 2002, Robinson College, University of Cambridge, UK . 2002

机译：体内生物素化的载脂蛋白：大肠埃希氏菌细胞超量生产
5. Engineering Fatty Acid Overproduction in Escherichia coli for Next-Generation Biofuels [D] . Lennen, Rebecca M. 2012

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6. Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports [O] . Benevides C. C. Pessela, Alejandro Vian, César Mateo, 2003

机译：Thermus sp。的生产过剩。大肠杆菌中的菌株T2β-半乳糖苷酶及其定制金属螯合物载体的制备
7. Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports [O] . Pessela, Benevides C. C., Vian, Alejandro, Mateo, César, 2003

机译：Thermus sp。的生产过剩。大肠杆菌中的菌株T2β-半乳糖苷酶及其定制金属螯合物载体的制备
8. Development of Sucrose-Fermenting Escherichia Coli K-12 Strains for Low- Cost Overproduction of Recombinant Proteins [R] . Lengyel, Z. 1999

机译：用于低成本过量生产重组蛋白的蔗糖发酵大肠杆菌K-12菌株的研制

Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

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