Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme.
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Kabashima T, Kitazono A, Kitano A, Ito K, Yoshimoto T
Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme.
J Biochem. 1997 Sep;122(3):601-5.
- PubMed ID
- 9348090 [ View in PubMed]
- Abstract
We cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.