scholarly journals Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli

2004 ◽  
Vol 385 (1) ◽  
pp. 181-187 ◽  
Author(s):  
Benedykt WLADYKA ◽  
Katarzyna PUZIA ◽  
Adam DUBIN
Keyword(s):  
Active Site ◽  
Genomic Dna ◽  
Specific Inhibitor ◽  
Insoluble Fraction ◽  
Immobilized Metal Affinity Chromatography ◽  
Expression Vectors ◽  
Native Conformation ◽  
Natural Sources ◽  
Metal Affinity ◽  
Genes Encoding

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.

scholarly journals A Cluster of Cuticle Protein Genes of Drosophila melanogaster at 65A: Sequence, Structure and Evolution

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1213-1224
Author(s):  
Jean-Philippe Charles ◽  
Carol Chihara ◽  
Shamim Nejad ◽  
Lynn M Riddiford
Keyword(s):  
Drosophila Melanogaster ◽  
Genomic Dna ◽  
Multiple Copy ◽  
Sequence Structure ◽  
Coding Regions ◽  
The Third ◽  
Genetic Complexity ◽  
Genes Encoding ◽  
Cuticle Protein ◽  
Cuticle Proteins

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.

The clp1 Gene of the Mushroom Coprinus cinereus Is Essential for A-Regulated Sexual Development

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 133-140
Author(s):  
Kazumi Inada ◽  
Yoshinori Morimoto ◽  
Toshihide Arima ◽  
Yukio Murata ◽  
Takashi Kamada
Keyword(s):  
Sexual Development ◽  
Genomic Dna ◽  
Mutant Allele ◽  
Coprinus Cinereus ◽  
Mating Partner ◽  
Essential Step ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Necessary And Sufficient ◽  
Novel Protein

Abstract Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

scholarly journals Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

2002 ◽  
Vol 184 (17) ◽  
pp. 4666-4671 ◽  
Author(s):  
Aaron J. Tooley ◽  
Alexander N. Glazer
Keyword(s):  
Ternary Complexes ◽  
Covalent Attachment ◽  
Heme Oxygenase 1 ◽  
Α Subunit ◽  
Ionization Time ◽  
E Coli ◽  
Metal Affinity ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

2010 ◽  
Vol 82 (1) ◽  
pp. 39-55 ◽  
Author(s):  
Rajasekar R. Prasanna ◽  
Mookambeswaran A. Vijayalakshmi
Keyword(s):  
Capillary Electrophoresis ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Immobilized Metal Affinity Chromatography ◽  
Metal Affinity ◽  
Purification Technique ◽  
Affinity Gel Electrophoresis ◽  
Insight Into ◽  
Histidine Tag

Immobilized metal-ion affinity (IMA) adsorption is a collective term that is used to include all kinds of adsorptions where the metal ion serves as the characteristic and most essential part of adsorption center. Of all the IMA techniques, immobilized metal-affinity chromatography (IMAC) has been gaining popularity as the choice of purification technique for proteins. IMAC represents a separation technique that is primarily useful for proteins with natural surface exposed-histidine residues and for recombinant proteins with engineered histidine tag. This review also gives insight into other nonchromatographic applications of IMA adsorption such as immobilized metal-ion affinity gel electrophoresis (IMAGE), immobilized metal-ion affinity capillary electrophoresis (IMACE), and immobilized metal-ion affinity partitioning (IMAP).

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