Naslov
Construction, cloning and expression of cystein enriched human microurokinase

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

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts / Flogel, Mirna - Zagreb : Jarža d.o.o., 2000, 76-76

Skup
Kongres hrvatskih biokemičara i molekularnih biologa uz međunarodno sudjelovanje HB2000

Mjesto i datum
Zagreb, Hrvatska, 13.10.2000. - 15.10.2000

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
mikrourokinaza; katalitička domena; ciljna mutageneza; proteinsko inženjerstvo; komparativno proteinsko modeliranje
(microurokinase; catalytic domain; site directed mutagenesis; comparative protein modelling)

Sažetak
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 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 with its activity. mu-PA 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 PA activity of expressed, partially pure mu-PA variants was measured using the esterolytic assay with the substrate Z-lys-thiobenzyl ester and the chromogen 5-5'-dithiobis-(2-nitrobenzoic acid). Both mu-PA and its cytein enriched variants, with the mutations E18(142)C, T28(152)C or L285(409)C as well as a combination of these residues, exserted plasminogen dependent PA (esterolytic) activity comparable to that of the wild type u-PA. As determined by comparative protein modelling, cystein substitution(s) did not cause measurable conformational changes in mu-PA with regard to its catalytic triad.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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