Avtor/Urednik		Bavec, Saša
Naslov		Kloniranje in ekspresija prve in tretje domene ekvistatina v Escherichia coli
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		1999
Obseg		str. 59
Jezik		slo
Abstrakt		Equistatin isolated from sea anemone Actinia equina, is a protein composed of three thyroglobulin-type 1 domains. It is an inhibitor of both papain-like cysteine proteinases and cathepsin D. Its structure and inhibitory activity suggest that equistatin belongs to thyropin family. We have developed procedure for the production of recombinant frst (Eq d-1 ) and third (Eq d-3) domains of equistatin. DNA fragments coding Eq d-1 or Eq d-3 were amplifed using PCR method and cloned into vector pUC 19, the nucleotide sequences of DNA fragments coding for equistatin domains were verified and further cloned into vector pET-22b(+) cas. The constructs pKG1 D and pKG3D enable the expression of the target proteins as fusion proteins with histidine hexapeptide on the N-terminus followed by a hexapeptide - a specific cleavage site for thrombin. The amino acid sequence of recombinant Eq d-1, as deduced from the nucleotide sequence, differs from the first domain of native equistatin by two amino residues and that for recombinant Eq d-3 differs from the third domain of the native protein by four residues. The E.coli BL21 (DE3) expression strain was transformed by pKG1 D or pKG3D constructs. Target proteins were expressed using the T7 expression system. Bacterial cells were disrupted by a combination of freeze/thaw cycles and sonication. Before the purification by Ni-NTA chromatography, high molecular weight, proteins were precipitated with ammonium sulfate. RP-HPLC was used as the fnal step in the purifcation procedure. The molecular weights of isolated recombinant Eq d-1 and Eq d-3 as determined by SDS-PAGE is 10 kDa.. CD spectra of both recombinant equistatin domains were also measured and both indicate aperiodic protein structure. By use of the described process for the preparation of recombinant Eq d-1 and Eq d-3 from one litre of bacterial culture approximately 0. (Abstract truncated at 2000 characters).
Deskriptorji		CNIDARIAN VENOMS CLONING, MOLECULAR ESCHERICHIA COLI RECOMBINANT FUSION PROTEINS TRANSFORMATION, GENETIC POLYMERASE CHAIN REACTION BASE SEQUENCE AMINO ACID SEQUENCE