Heterologous expression of human granzyme K in Bacillus subtilis and characterization of its hydrolytic activity in vitro.

Human granzyme K, a serine protease found in secretory granules of cytotoxic T-lymphocytes, was produced in its catalytically active form by recombinant technology using Bacillus subtilis as host. The enzyme displays 40-45% identity to other members of the human granzyme group, and its closest homologue (75% identity) is the rat tryptase RNK-tryp2. The recombinant protein can be recovered in its mature form from the bacterial culture supernatant and purified by cation exchange chromatography. Initial characterization reveals a protein of approximately 28 kDa that is specifically labelled by [3H]di-isopropyl fluorophosphate. Measurements of Kcat/K(m) for single-residue thioester substrates show approximately a two-fold preference for a Lys versus Arg residue at Pl. No activity was observed on ester substrates with various other residues at the Pl position. Using oligopeptide substrates, the enzyme displays peptidolytic activity C-terminal to both Lys and Arg residues with comparable rates of hydrolysis. Likewise, substrate hydrolysis is blocked most efficiently by inhibitors that contain Lys or Arg at position Pl. The availability of the cloned enzyme will facilitate the analysis of biological roles for this novel granzyme, and differentiate its activity from that of other granzymes.

Cloning of cDNA for human granzyme 3.

A serine protease gene was cloned from cDNA prepared from tissue isolated from human ascites. The gene codes for a protein of 264 amino acids, identified as human granzyme 3 by the N-terminal amino acid sequence. Granzyme 3 has the expected features of the chymotrypsin family of serine proteases, and is closely related to other human granzymes (40-45% identity). However, the closest granzyme 3 homologue is the recently characterized rat tryptase, RNK-Tryp-2 (75% identity). From Northern blots, granzyme 3 appears to be highly expressed in peripheral blood leukocytes, spleen, thymus, and lung tissues.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells.

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Two histidines are essential for the activity of the beta-galactosidase from Lactobacillus delbrückii subsp. bulgaricus.

Twelve tyrosines, ten glutamates, and five histidines were individually substituted for phenylalanine, glutamine, and asparagine, respectively, in the beta-galactosidase from Lactobacillus delbrückii subsp. bulgaricus. Only Y509, E464, H351, and H534 appear to be essential for the activity of the enzyme. The residues Y509 and E464 are homologous to Y503 and E461, respectively, of the Escherichia coli lacZ beta-galactosidase which have been shown to be necessary for its activity. Surprisingly, a number of amino acids that are highly conserved in the sequence alignments of nine beta-galactosidases and four beta-glucuronidases are not essential for enzyme activity.

Generation and Characterization of Environmentally Sensitive Variants of the beta-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus.

A method is described for generating and screening variants of the beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the beta-galactosidase gene carried on an Escherichia coli expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on beta-galactosidase indicator plates. The mutations responsible for three variant beta-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.

Characterization of two cold-sensitive mutants of the beta-galactosidase from Lactobacillus delbruckii subsp. bulgaricus.

Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli.

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.