Recombinant maize protoporphyrinogen IX oxidase expressed in Escherichia coli forms complexes with GroEL and DnaK chaperones.

The clone corresponding to maize plastidic protoporphyrinogen IX oxidase (PPO) has been isolated by functional complementation and inserted into a pET16b vector for expression in Escherichia coli. Recombinant PPO was purified by standard affinity chromatography using a metal chelating resin. Two contaminants copurified with recombinant PPO and were identified as GroEL and DnaK. Since chaperone binding to hydrophobic regions of the protein is regulated by ATP availability, an ATP washing step was introduced prior to elution of the recombinant protein from an affinity column. This washing step selectively removed both chaperones and allowed the recovery of pure PPO. Coexpression of PPO and GroELS resulted in a sixfold increase of soluble PPO yield, suggesting that bacterial chaperones could be limiting during the folding of the heterologous protein. However, a portion of PPO was still found in the insoluble fraction. Buffer containing the GroEL and DnaK enabled resuspension of PPO from the insoluble fraction but failed to enhance refolding of the denaturated protein. Attempts to increase the amount of soluble PPO using a thioredoxin-PPO fusion protein were not successful. Initial characterization of the recombinant PPO found that it possessed a high V(max), an elevated affinity for substrate, and an elevated sensitivity to PPO inhibitor herbicides compared to previous reports.

NMR solution structure of the pathogenesis-related protein P14a.

The nuclear magnetic resonance (NMR) structure of the 15 kDa pathogenesis-related protein P14a, which displays antifungicidal activity and is induced in tomato leaves as a response to pathogen infection, was determined using 15N/13C doubly labeled and unlabeled protein samples. In all, 2030 conformational constraints were collected as input for the distance geometry program DIANA. After energy-minimization with the program OPAL the 20 best conformers had an average root-mean-square deviation value relative to the mean coordinates of 0.88 A for the backbone atoms N, C(alpha) and C', and 1.30 A for all heavy atoms. P14a contains four alpha-helices (I to IV) comprising residues 4 to 17, 27 to 40, 64 to 72 and 93 to 98, a short 3(10)-helix of residues 73 to 75 directly following helix III, and a mixed, four-stranded beta-sheet with topology +3x, -2x, +1, containing the residues 24-25, 53 to 58, 104 to 111 and 117 to 124. These regular secondary structure elements form a novel, complex alpha + beta topology in which the alpha-helices I, III and IV and the 3(10)-helix are located above the plane defined by the beta-sheet, and the alpha-helix II lies below this plane. The alpha-helices and beta-strands are thus arranged in three stacked layers, which are stabilized by two distinct hydrophobic cores associated with the two layer interfaces, giving rise to an "alpha-beta-alpha sandwich". The three-dimensional structure of P14a provides initial leads for identification of the so far unknown active sites and the mode of action of the protein, which is of direct interest for the generation of transgenic plants with improved host defense properties.

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits.

To test the potential usefulness of transgenic rabbits as production systems for human proteins of pharmaceutical value, we cloned the rabbit beta-casein promoter and fused it to the genomic sequence of the human interleukin-2 (hIL2) gene. Four transgenic female rabbits were tested for expression and biological activity of the foreign protein in their milk. The milk of all four females proved to contain biologically active hIL2. The results show that transgenic rabbits may represent a convenient and economic system for the rapid production of biologically active protein in milk.

Cloning of the saliva-interacting protein gene from Streptococcus mutans.

Genomic libraries from Streptococcus mutans OMZ175 were constructed in bacteriophage vectors. DNA fragments 1 to 2 kilobases in length were cloned in expression vector lambda gt11. S. mutans DNA fragments 15 to 20 kilobases in length were inserted in the BamHI site of phage EMBL3. Rabbit antiserum raised against an S. mutans saliva-interacting protein with a molecular weight of 74,000, designated 74K SR, was used to screen the lambda gt11 library. A recombinant phage carrying an S. mutans DNA sequence of 1.45 kilobases, lambda SmAD2, was detected and isolated. This fragment, named SmAD2, was used to construct the recombinant expression plasmid pSAD2-4 which encoded for the expression of a 60,000-molecular-weight protein controlled by the beta-galactosidase promoter from plasmid pUC8. The SmAD2 fragment and polyclonal anti-74K SR antibodies were used to screen the EMBL3 library. A total coincidence between the screening with antibodies and the DNA probe was observed, and two phages, lambda SmAD9 and lambda SmAD10, were isolated. They contained a common S. mutans DNA sequence of about 11.8 kilobases and coded for a protein with a molecular weight of about 195,000, which comigrated with a protein of an S. mutans cell wall extract. The expressed protein was purified, and a very strong relationship with the S. mutans 74K SR protein was found by competitive enzyme-linked immunosorbent assay. Thus, cloning of the 74K SR gene allowed us to demonstrate that the saliva receptor appears to be a part of an S. mutans precursor molecule with a molecular mass of 195,000 daltons.

Local response in rat to liposome-associated Streptococcus mutans polysaccharide-protein conjugate.

The effect of gastric intubation with soluble or liposome-associated Streptococcus mutans serovar polysaccharide, 74-kDa saliva receptor (74K SR protein) or polysaccharide-74K SR protein conjugate on the locally induced salivary IgA response and memory in rats was investigated. Animals immunized on four successive days with soluble antigens showed a weak salivary anti-74K SR protein or anti-polysaccharide IgA response. Rats primed and boosted by a single injection of liposome-associated 74K SR protein or polysaccharide-74K SR protein conjugate developed a salivary anti-74K SR protein IgA and IgG primary and secondary response. A primary anti-polysaccharide response was only observed in saliva of animals immunized with either high concentration of liposome-associated polysaccharide or liposome-associated polysaccharide-74K SR protein conjugate. However, a secondary local anti-polysaccharide IgA response was detected in animals boosted with liposome-polysaccharide-74K SR protein conjugate. No such anamnestic response was seen when high dose of liposome-associated polysaccharide was used to boost the animals. Furthermore, the salivary anti-polysaccharide IgA response paralleled the anti-74K SR protein IgA response. These studies showed that intragastric immunization of rats with liposome-associated polysaccharide-74K SR protein conjugate produced a local anti-polysaccharide IgA memory.