MAB1012 Sigma-AldrichAnti-Alkaline Phosphatase Antibody, E. coli, bacterial only

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

The BceB protein of the cystic fibrosis mucoid isolate Burkholderia cenocepacia IST432 is proposed to catalyze the first step of the exopolysaccharide repeat unit assembly. Extracts of Escherichia coli cells overexpressing BceB were shown to contain glycosyltransferase activity and mediate incorporation of glucose-1-phosphate into membrane lipids. The amino acid sequence of BceB exhibits two conserved regions, one comprising two invariant aspartic acid residues (Asp339 and Asp355) that are essential for catalysis, as substantiated by site-directed mutagenesis, and the other comprising a putative Rossmann fold motif. The results of protein topology analysis using PhoA and LacZ fusions supported in silico predictions that BceB has at least six transmembrane segments and two major cytoplasmic loops comprising the conserved regions described above.

SETTING: Mycobacterium avium is the major cause of disseminated infection in patients with late stage AIDS.Objective: In order to identify M. avium genes that may be involved in bacterial uptake and intracellular survival, a phoA -based reporter system was used to identify genes that encoded surface-expressed or exported proteins. DESIGN: PhoA (alkaline phosphatase) is only active if the protein is exported across the cell membrane into the periplasm. Consequently, detectable PhoA activity requires the fusion of a promoterless phoA gene with a DNA fragment containing a functional promoter and export leader sequence. A M. avium promoter library was constructed in the phoA reporter plasmid pJEM11 and screened in M. smegmatis for expression of active PhoA. RESULTS: More than 100 independent PhoA(+)recombinants were isolated, of which 15 were sequenced. Most of these exhibited varying degrees of homology with published M. avium, M. tuberculosis, M. bovis and M. leprae sequences. Based on sequence homology, one M. avium sequence was identified as a homologue of the M. tuberculosis phosphate transport gene phoS2 (Ag88). Another M. avium sequence was homolog with a putative M. tuberculosis cutinase gene. Both of these M. avium genes were cloned and sequenced. Several other M. avium sequences were homologous with, as yet, unidentified M. tuberculosis genes. CONCLUSION: PhoA fusion technology is applicable to the study of atypical slow growing mycobacteria. Most of the M. avium exported proteins identified in this study are highly homologous with genes from M. tuberculosis and M. leprae. In addition, parallels in gene organization were identified between M. avium and members of the M. tuberculosis complex.

Proteins translocated across the single plasma membrane of mycoplasmas (class Mollicutes) represent important components likely to affect several interactions of these wall-less microbes with their respective hosts. However, identification and functional analysis of such proteins is hampered by the lack of mutational systems in mycoplasmas and by a perceived limitation in translating recombinant mycoplasma genes containing UGA (Trp) codons in other eubacteria. Here we directly analyze a gene encoding a Mycoplasma hyorhinis protein capable of promoting its membrane translocation. It was initially detected by screening a recombinant phage genomic library with antibody from a host with M. hyorhinis-induced arthritis and was localized by Tn5 and deletion mutations affecting expression of antigenic translational products. Sequence analysis of the isolated gene predicted a hydrophilic protein, P101, containing three UGA codons and a putative signal peptide with an uncharacteristic cluster of positively charged amino acids near its C terminus. Nevertheless, lambda::TnphoA transposon mutagenesis of an Escherichia coli plasmid bearing the p101 gene resulted in p101::TnphoA fusions expressing products that could translocate as much as 48 kDa of the P101 sequence (up to the first UGA codon) across the E. coli plasma membrane. Fusion proteins containing mature P101 sequences expressed mycoplasma epitopes and were found by cell fractionation and detergent phase partitioning to be integral membrane proteins in E. coli, suggesting a lack of signal peptide cleavage in this system. Importantly, identification of P101 by direct analysis of its export function relied neither on prior identification of the mycoplasmal product nor on complete expression of the product from the cloned mycoplasma gene.

Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.

Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.

We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.

We have constructed a series of plasmids containing a modified form of the phoA gene of Escherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme beta-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.