title: Isolation and bioinformatic analysis of seven genes encoding potato apyrase. Bacterial overexpresssion, refolding and initial kinetic studies on some recombinant potato apyrases.
abstract: Here we have isolated seven apyrase encoding cDNA sequences (StAPY4-StAPY10) from the potato variety Saturna tuber cDNA library by affecting necessary modifications in the screening protocol. The cDNA sequences were identified with a pair of primers complementary to the most conserved sequences identified in potato variety Desiree apyrase genes. Our data strongly suggest the multigenic nature of potato apyrase. All deduced amino acid sequences contain a putative signal sequence, one transmembrane region at the amino terminus and five apyrase conserved regions (ACRs) (except StAPY6). Phylogenetic analysis revealed that encoded proteins shared high level of DNA sequence identity among themselves, representing a family of proteins markedly distinct from other eukaryotic as well as prokaryotic apyrases. Two cDNA sequences (StAPY4 and StAPY6) were overexpressed in bacteria and recombinant proteins were found accumulated in inclusion bodies, even thought they were fused with thioredoxin-tag. Additionally, we present the first successful in vitro attempt at reactivation and purification of recombinant potato apyrase StAPY6. The ratio of ATPase/ADPase hydrolysis of recombinant StAPY6 was determined as 1.5:1. Unlike other apyrases the enzyme lacked ACR5 and was endowed with lower molecular weight, high specificity for purine nucleotides and very low specificity for pyrimidine, suggesting that StAPY6 is a potato apyrase, not described so far.
authors.affiliation: Department of Biochemistry, Faculty of Biology and Environment Protection, Nicolaus Copernicus University, Lwowska 1 St, 87-100 Toruń, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wujak
authors.forename: Mariusz
authors.initials: M
authors.lastname: Banach
authors.forename: Dorota
authors.initials: D
authors.lastname: Porowińska
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Piskulak
authors.forename: Michał
authors.initials: M
authors.lastname: Komoszyński
mesh_terms: D001081:Apyrase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D001081:Apyrase
keywords: Apyrase; Bacterial overexpression; Bioinformatic analysis; NTPDase; Refolding; cDNA library screening