Author/Editor		Galeša, Katja
Title		Ekspresija, izolacija in karakterizacija ekvistatina in posameznih domen
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2002
Volume		str. 108
Language		slo
Abstract		Equistatin is a protein isolated from the sea anemone Actina equina. It is composed of three thyroglobin type-1 domains and is a member of thyropins, a group of protease inhibitors with thyroglobulin type-1 domains. The members of thyropins (saxiphilin, p41, and ECI) are all inhibitors of papain-like cysteine proteases. In addition, equistatin displays inhibitory activity against an aspartic protease, cathepsin D. The full-length cDNAs coding for equistatin and for its individual domains were inserted in the expression vectors pKG1 or pET22b(+). The nucleotide sequence of the inserts in the vectors was determined. Recombinant proteins were expressed in E. coli BL21(DE3) with His-tag fused to either the N- or the C- terminus. The proteins were purified by Ni-NTA affinity chromatography, and their identities verified via Nterminal sequencing. The final yield of purified proteins was approximately 1 mg of protein per I of bacterial culture. Because of the relatively low yield from the bacterial expression system, the proteins were expressed in the yeast P. pastoris. cDNAs for individual protein domains were cloned into a yeast expression vector pPIC9. After determining the nucleotide sequence of the inserts, the DNA was linearized with SaII and transformed into P. pastoris by electroporation. The highest expression level clones were selected and recombinant domains were produced in a fermenter. The expression levels of equistatin domains in P, pastoris were about 1 g/I. The recombinant proteins were isolated by hydrophobic chromatograpžy on Phenyl-Sepharose and gel filtration. Nterminal analyses of domains showed four additional aminoacids, EAEA, indicating incomplete removal of the a-mating factor preprosignal. (Abstract truncated at 2000 characters).
Descriptors		CNIDARIAN VENOMS TRANSFECTION TRANSFORMATION, GENETIC RECOMBINANT FUSION PROTEINS DNA, RECOMBINANT ESCHERICHIA COLI YEASTS BACTERIOPHAGE M13 CATHEPSINS PAPAIN SEQUENCE ANALYSIS, DNA BASE SEQUENCE SEA ANEMONES