Naslov
Construction, cloning and expression of cystein enriched human microurokinase.

Autori
Brdar, Branko ; Rubelj, Ivica ; Čović, Marcela ; Reich, Edward

Izvornik
Periodicum biologorum (0031-5362) 101 (1999), 4; 311-318

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
microurokinase; urokinase-type plasminogen activator; protein engineering; comparative protein modelling
(protein engineering; comparative protein modelling)

Sažetak
Background and purpose: Urokinase-type plasminogen activator (u-PA), a trypsin-like serine protease, is a glycoprotein composed of an epidermal growth factor-like (EFG), a kringle and a serine protease domain. The aim of this study was to construct and express a truncated enzymatically active form of urokinase, microurokinase (mu-PA), consisting essentially of the protease domain, as well as to introduce in the sequence of this two-domain less structure several cystein residues that would not interfere (affect) with its activity. Material and methods: Microurokinase and its cystein substituted variants were engineered from full-length u-PA cDNA using PCR and oligonucleotide primers of appropriate sequence. mu-PA and its variants were cloned and expressed in the baculovirus and Pichia pastoris expression systems, respectively. Plasminogen dependent plasminogen activator activity of expressed and secreated, partially pure muPA variants was measured using the esterolytic assay with the substrate Z-lys-thiobenzyl ester and the chromogen 5-5'-dithiobis-(2-nitrobenzoic acid). Results and conclusions: Both microurokinase and its cystein enriched variants exerted plasminogen dependent plasminogen activator (esterolytic) activity comparable to that of the wild type u-PA. As determined by comparative protein modelling using the coordinates of the crystal structure of low molecular weight uPA retreivable from the Protein Data Bank, cystein substitution(s) did not cause measurable conformational changes in mu-PA with regard to its catalytic triad.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA