title: Efficient solubilization, purification of recombinant extracellular alpha-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli.
abstract: The gene encoding the Pyrococcus furiosus extracellular alpha-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant alpha-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90 degrees C treatment for 3 min in Britton-Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher than previously reported.
authors.affiliation: Shanghai Institutes for Biological Sciences, Chinese Academy of Science, 500 Caobao Road, 200233 Shanghai, China.
authors.forename: Lisa
authors.initials: L
authors.lastname: Wang
authors.forename: Qinhua
authors.initials: Q
authors.lastname: Zhou
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Chu
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Lu
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shengli
authors.initials: S
authors.lastname: Yang
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases