Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in <i>E. coli</i> as fusion protein and its isolation

Auerswald, Ennes A.; Genenger, Gabriele; Assfalg-Machleidt, Irmgard; Kos, Janko; Bode, Wolfram

doi:10.1016/0014-5793(89)80127-9

Cited by 19 publications

(10 citation statements)

References 31 publications

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“…The constructions of AV55, AV55-$56, AP103-L105, AI102-QI07 and loop2-KD2 were performed by cassette mutagenesis using the synthetic cystatin gene. Briefly, the large Xbal-PstI fragment of pGG 8.1.1, [10] was substituted by appropriate deleted DNA sequences for AV55 and AV55-$56. The large DralII-XhoI fragment was substituted by fragments with deletions for AP103-L105 and AI102-Q107 or with the fragment for loop2-KD2 having the sequence 5' g tgc acc ttc gtt gtt tac ATC GAC ATC CAG CTG CGT ATC GCT aaa ctt c 3' (capital letters indicate the substituted nucleotides).…”

Section: Molecular Cloning Cassette Mutagenesis and Expression Of Thementioning

confidence: 99%

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Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

et al. 1995

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~. IntroductionChicken cystatin, a small protein-type inhibitor of cysteine proteinases, represents a well studied member of the cystatin ,uperfamily (for reviews see [2,3]). It was shown to be a reversble tight-binding inhibitor of papain and papain-like enzymes 4Corresponding author. Fax: (49) (89) 5160 4735. E-mail: tbbreviations. E-64, t.-3-carboxy-2,3-trans-epoxy-propionyl-leucyl~Lmido-(4-guanidino)butane; NH-Mec, 7-(4-methyl) coumaryl-amide; . [4-9]. Synthetic genes of chicken cystatin and variants of them have been expressed and characterized [10][11][12]. Crystal and solution structures of native and recombinant chicken cystatin are known [13,14] and a model of the interaction of chicken cystatin with papain has been proposed [7,13]. According to the so-called 'elephant-trunk model', the enzyme-inhibitor complex is formed mainly by hydrophobic interactions between chicken cystatin and the complementary active-site cleft of papain, whereby cystatin contributes the N-terminal 'trunk' (Leu7-Gly9), the first hairpin loop (Gln53-Gly57) and the second hairpin loop (Pro]°3-Leul°5). The validity of this model was confirmed by elucidation of the structure of the complex formed between S-carboxymethylated papain and stefin B [15] as well as by kinetic data obtained for the interactions of native and/or modified cystatins with different cysteine proteinases [7,8,12,16].Detailed analyses of the N-terminal segments of chicken cystatin and human cystatin C indicate a substrate-like interaction of this region with residues of the subsites $3 and $2 of the proteinase. Especially the amino acid residues preceding the conserved Gly 9 of chicken cystatin or Gly ~1 of human cystatin C have distinct but different effects on the binding to papain, actinidin, ficin, cathepsin B or cathepsin L [17 20].The contribution of the first hairpin loop of cystatins to complex formation has not yet been investigated in such detail. From studies with KVVAG-and QVTAG-mutants of cystatin A, Nikawa et al. [21] proposed that the first hairpin loop is not essential for cysteine proteinase inhibition. But the importance of this region is strongly suggested by (i) the structural data of the crystallized stefin B-papain complex [15], (ii) its conserved primary structure (QxVxG-region) [2] and (iii) inhibition properties measured with substitution variants of oryzacystatin [22] and chicken cystatin [12]. The latter data provide first evidence that amino acid residues of this region are partially responsible for the distinct affinities of cystatins for different cysteine proteinases. However, no data have been presented so far on variants of the first hairpin loop missing conserved amino acid residues.Little is known about the importance of the second hairpin loop for the stability of the molecule itself or its contribution to complex formation. Using spectroscopic methods, Lindahl and co-workers [6] demonstrated that Trpl°4 of chicken cystatin must be involved in the interaction with papain. These early data...

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Section: Molecular Cloning Cassette Mutagenesis and Expression Of Thementioning

confidence: 99%

“…[4][5][6][7][8][9]. Synthetic genes of chicken cystatin and variants of them have been expressed and characterized [10][11][12]. Crystal and solution structures of native and recombinant chicken cystatin are known [13,14] and a model of the interaction of chicken cystatin with papain has been proposed [7,13].…”

Section: ~ Introductionmentioning

confidence: 99%

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

et al. 1995

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“…The family 2 cystatins that have been studied include (1) a chemically synthesized human cystatin C gene (Strauss et al, 1988); (2) an authentic cystatin C cDNA Dalb0ge et al, 1989); and (3) a synthetic gene-encoding chicken egg white cystatin (Auerswald et al, 1989). The family 1 cystatins include a chemically synthesized human cystatin A gene (Kaji et al, 1989 and1990) and a synthetic gene-encoding rat cystatin a (Katunuma et al, 1988).…”

Section: Status Of Cystatin Expression In E Colimentioning

confidence: 99%

Cystatins — Inhibitors of Cysteine Proteinases

Bobek

Levine

1992

Critical Reviews in Oral Biology & Medicine

146

100

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ABSTRACT:The cystatin superfamily of proteins, derived from a common ancestor, is comprised of a diverse group of potent cysteine proteinase inhibitors and antibacterial/viral agents grouped into several families. This review concentrates on family 2 cystatins, namely, the human salivary cystatins and cystatin C. Emphasis is given to their physicochemical and functional properties at both the protein and the molecular level. The role of cystatins in disease processes, including those in the oral cavity, is also discussed. Finally, future directions for cystatin research in oral biology are presented.

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“…The deletions of the desired sequences were performed with the vector pGG 8.1.1 [27] harbouring the synthetic Arg-Glu-Phe-[Metl, Ile29, Leu891-chicken cystatin gene (data library number, EMBL X14685) and the variants were obtained by cassette mutagenesis [28]. After restriction digestion of the vector with EcoRUSucI, the fragment coding for the N-terminal extension was deleted from the pGG 8.1.1.…”

Section: Cassette Mutagenesis Molecular Cloning and Expression Of Thmentioning

confidence: 99%

Production, Inhibitory Activity, Folding and Conformational Analysis of an N‐terminal and an Internal Deletion Variant of Chicken Cystatin

Auerswald

Nägler

Schulze

et al. 1994

European Journal of Biochemistry

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Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Metl, Ile29, Leu891-chicken egg white cystatin gene in Escherichia coli. The variant des-Serl -Proll-[Alal2, Glu13, Phel4, Metl5, Ile29, Leu891-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Metl, Ile291-des-Cys71 -Met89-chicken cystatin (del-helix 11) were purified and characterized by inibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix I1 variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix I1 variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing GddHC1 concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral PI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100000-fold higher than those obtained with natural and recombinant chicken cystatin.Chicken cystatin is a small protein inhibitor (116 amino acids) of cysteine proteinases [l, 21 and the best studied member of the cystatin superfamily [3,4]. It has been shown that this molecule is a reversible, tight-binding inhibitor of papain-like enzymes [5-81. A model for the interaction of papain with chicken cystatin was derived from X-ray crystallography of a N-terminally truncated chicken cystatin [91 and from kinetic studies [6, 71. Recently, the solution structures of natural phosphorylated chicken cystatin and of a recombinant variant, Arg-GluPhe-[Metl, Ile29, Leu891-chicken cystatin, were solved by

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Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

Cited by 19 publications

References 31 publications

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

Cystatins — Inhibitors of Cysteine Proteinases

Production, Inhibitory Activity, Folding and Conformational Analysis of an N‐terminal and an Internal Deletion Variant of Chicken Cystatin

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