ABSTRACT
A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.
Subject(s)
Cystatins/genetics , Skin/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Base Sequence , Binding Sites/genetics , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/genetics , Female , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Recombinant Proteins/geneticsABSTRACT
Clinically-isolated methicillin-resistant Staphylococcus aureus (MRSA) strain 743 exhibited resistance to tetracycline as judged from the active efflux of the drug. The efflux of tetracycline was inhibited by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and minocycline. Inhibitors of the efflux pump were examined in this strain to determine the cellular accumulation of tetracycline. Out of seven compounds examined, three caused a significant increase in the cellular concentration of tetracycline by inhibiting the efflux pump. Two of them seem to be energy inhibitors. Ro 07-3149 inhibited the efflux pump without affecting the energy state, and exhibited very low antibacterial activity but showed weak synergy with tetracycline.
Subject(s)
Staphylococcus aureus/drug effects , Tetracycline/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biological Transport , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Humans , Indans/pharmacology , Membrane Proteins/genetics , Microbial Sensitivity Tests , Minocycline/pharmacology , Staphylococcus aureus/isolation & purification , Staphylococcus aureus/metabolism , Tetracycline/pharmacology , Tetracycline Resistance , Uncoupling Agents/pharmacologyABSTRACT
A novel indan derivative, 1,1-dimethyl-5-(1-hydroxypropyl)-4,6,7-trimethylindan (Ro 07-3149), was found to be a strong inhibitor of the tet(K) gene-encoded tetracycline/H+ antiporter of Staphylococcus aureus. One micromole of this compound per mg membrane protein was enough for complete inhibition of the Tet(K)-mediated tetracycline transport and tetracycline-coupled proton transport, without the energy state of the membrane being affected. The mode of inhibition was non-competitive. Although this compound caused membrane de-energization at a high concentration, the IC50 value for de-energization (7.3 micromol/mg membrane protein) was about 17 times and 33 times higher than the values for Tet(K)-mediated proton/tetracycline antiport and [3H]tetracycline transport, respectively, indicating that the inhibitory action of Ro 07-3149 is not due to the uncoupling effect of the inhibitor.