Avtor/Urednik		Drobnič, K
Naslov		Kloniranje in ekspresija gena za steroidno 1,2-dehidrogenazo bakterije Nocardia opaca
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		1995
Obseg		str. 61
Jezik		slo
Abstrakt		Steroids are growth substrates for a wide variety of microorganisms. The degradation of steroids begins with the introduction of a double bond into ring A. Steroid 1,2-dehydrogenase is the key enzyme in the degradation of steroids. The introduction of the double bond between carbon atoms 1,2 in the steroid ring A is essential in the production of glucocorticoids analogs. Analogs modified in this way have diminish side effects. This enzyme is also of great importance for the production of various glucocorticoid analogs or steroids with a phenolic A-ring (e.g. estrone). The clone pSTD23, which hybridized with the probe designed according to the N-terminal amino acid sequence of the protein, was restricted into smaller fragments. Overlapping DNA fragments were generated by subcloning of appropriate restriction fragments into pUC19 vector as well as by unidirectional nested deletions using both exonuclease III and nuclease s1. The gene for steroid 1,2-dehydrogenase was found onto two fragments: the 0.7 kb Pstl/Kpnl and the 2.6 kb Kpnl fragment respectively. Both of them were subcloned into pUC19 and three transformed colonies having correct orientation was found. The enzymatic activity was 0.3 U/mg that is 100X lower than those obtained from induced cells of Nocardia opaca in semiquantitative tests. This finding however was expected, because the plasmid expressed 1,2-dehydrogenase gene product was of similar size on PAGE as the original protein, that is 56 kDa. The complete sequence of steroid 1,2-dehydrogenase was determined. An open reading frame which encodes a 55.750 Da polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The amino acid sequence was found to be similar to the two already reported steroid 1,2-dehydrogenases from Pseudomonas testosteroni and Arthrobacter simplex as well as to the fumarate reductase of Shewanella putrefaciens.(trunc.)
Deskriptorji		NOCARDIA OXIDOREDUCTASES GENE EXPRESSION REGULATION, BACTERIAL CLONING, MOLECULAR DNA, RECOMBINANT ANDROSTENEDIONE ANDROSTADIENES GENOMIC LIBRARY ESCHERICHIA COLI BASE SEQUENCE