Kim, YK Choi, IG Nam, W Yu, YG 2024-01-21T13:09:45Z 2024-01-21T13:09:45Z 2021-09-05 2000-11-30 1225-8687 https://pubs.kist.re.kr/handle/201004/140918 Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa, Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I,-G,, Bang, W,-K,, Kim, S.-H., Yu, G, Y,, J, Biol. Chem. (1999), Vol. 274, pp, 881-888], Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E, coli, Also, the proteolytic activity of the expressed protein appeared to toxic to E, coli, An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purifiied to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around 90 degreesC using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface. English SPRINGER-VERLAG SINGAPORE PTE LTD METHANOCOCCUS QUANTITIES Identification of a mature form and characterization of thermostability of a serine-type protease from Aquifex pyrophilus Article 1 JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, v.33, no.6, pp.493 - 498 JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY 33 6 493 498 scie scopus 000165575200009 2-s2.0-0034357010 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article METHANOCOCCUS QUANTITIES Aquifex pyrophilus hyperthermophile protease thermostability expression