A gene fusion system for generating antibodies against short peptides

Löwenadler, Björn; Jansson, Birger; Paléus, Sven; Holmgren, Erik; Nilsson, Björn; Moks, Tomas; Palm, Gunnar; Josephson, Staffan; Philipson, Lennart; Uhlén, Mathias

doi:10.1016/0378-1119(87)90032-1

Cited by 121 publications

(65 citation statements)

References 21 publications

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“…The pEcC63ALpxC expression plasmid encoding E. coli LpxC with the C63A mutation (18) was modified to encode a fusion protein between LpxC and an IgG-binding protein, termed a ZZ tag (33)(34)(35), with a tobacco etch virus protease site in the linker region between the two proteins. An NcoI restriction site was introduced at the C-terminal stop codon of LpxC by using the QuikChange mutagenesis kit (Stratagene) with the primer 5Ј-CCT TCA GCT GTA CTG GCA CCA TGG GGA TCC GAA TTC GAG CTC CG-3Ј and its reverse complement.…”

Section: Methodsmentioning

confidence: 99%

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Gattis

Hernick

Fierke

2010

Journal of Biological Chemistry

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UDP-3-O-((R)To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/ zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K D values of 60 ؎ 20 pM and 110 ؎ 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.Gram-negative bacteria are important targets in the continuing fight against antibiotic-resistant infections. The development of new antibiotics to treat infections caused by resistant organisms is critically needed and will require novel targets. One potential source of targets in Gram-negative bacteria is the lipid A biosynthetic pathway (Fig. 1A) (1, 2), an essential building block of lipopolysaccharides (LPS) that make up the outer leaflet surrounding the cell wall in Gram-negative bacteria (2-4). In addition to being essential for cell viability, LPS are also referred to as endotoxins (2, 4, 5) and are responsible for immunogenic stimulation in septic shock that can lead to a number of deleterious effects, including severe hypotension, multiple organ dysfunction, and death (5). Consequently, inhibitors of lipid A biosynthesis have the potential to serve as both antibiotics and antiendotoxins (6). The UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) 3 catalyzes the committed and second overall step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine to UDP-3-O-myristoyl-glucosamine and acetate ( Fig. 1) (7-10). Consequently, inhibitors of LpxC are an active area of drug development (11)(12)(13)(14).LpxC was previously identified as a mononuclear Zn(II) metalloenzyme (Fig. 1B) (15) that also contains a weaker, inhibitory metal ion-binding site. This catalytic metal ion binds and polarizes an inner sphere water molecule that is activated by general base catalysis to serve as the nucleophile for hydrolysis of the acetyl substrate (Fig. 1C) (16, 17). However, recent evidence has shown that LpxC is more active with Fe(II) as its metal ion cofactor compared with Zn(II) (18). This increase in activity may correlate with a change in the ground stat...

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Section: Methodsmentioning

confidence: 99%

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Gattis

Hernick

Fierke

2010

Journal of Biological Chemistry

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“…A second was directed against the C-terminus of the 90-kDa chain of plasma factor VIII. A peptide conjugate containing a peptide identical with the amino acid sequence Tyr729 to Arg740 was produced biosynthetically in Escherichia coli expressing the peptide attached to the ZZ segment of protein A from Staphylococcus aureus (Lowenadler et al, 1987). The peptide conjugate was purified by IgG-affinity chromatography prior to immunization.…”

Section: Methodsmentioning

confidence: 99%

Novel Forms of B‐Domain‐Deleted Recombinant Factor VIII Molecules

Lind¹,

Larsson²,

Spira³

et al. 1995

European Journal of Biochemistry

123

111

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Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

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“…These experiments suggest that BacMPbased immunoassay systems have superior advantages due to their high sensitivity and rapid measurement of samples. The Z domain of protein A in Staphylococcus aureus has the ability to bind to the Fc region of IgG (Lowenadler et al 1987). A technique for the assembly of the Z domain on the BacMP surface by gene fusion using a protein A-magA hybrid gene was constructed ( Matsunaga et al 1999).…”

Section: Immunoassay and Receptor-binding Assaymentioning

confidence: 99%

Formation of magnetite by bacteria and its application

Arakaki

Nakazawa

Nemoto

et al. 2008

J. R. Soc. Interface.

220

176

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Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein-magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.

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A gene fusion system for generating antibodies against short peptides

Cited by 121 publications

References 21 publications

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Novel Forms of B‐Domain‐Deleted Recombinant Factor VIII Molecules

Formation of magnetite by bacteria and its application

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