Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.

Macferrin, Kurtis D.; Terranova, Michael P.; Schreiber, Stuart L.; Verdine, Gregory L.

doi:10.1073/pnas.87.5.1937

Cited by 99 publications

(64 citation statements)

References 52 publications

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“…polyphemus antennal PBP Apo-3 (Raming et al, 1989) were used for ECPCR ( Fig. 1) (MacFerrin et al, 1990). The N-terminal primer, a 55-mer, had an Eco RI site, an AAGGAG ribosome binding site, an ATG start codon, and codons for Ser' to Ser'.…”

Section: Resultsmentioning

confidence: 99%

“…In order to scale up protein expression and to obtain substantial quantities of isotopically labeled protein for 3D NMR analysis, a bacterial expression system was required. This paper describes the use of expression cassette polymerase chain reaction (MacFerrin et al, 1990) to engineer a bacterial overproducer (Schreiber & Verdine, 1991) of the A . polyphemus Apo-3 PBP, and documents that both the soluble and refolded recombinant PBP are indistinguishable from the native PBP isolated from moth antennae.…”

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confidence: 99%

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Bacterial expression and photoaffinity labeling of a pheromone binding protein

Prestwich

1993

Protein Science

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The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl 6-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave >95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E, 11Zhexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure.Keywords: Antheraea polyphemus; expression cassette PCR; Lepidoptera; olfactory signal transduction; pheromone binding protein; pheromone perception; photoaffinity labeling; protein refoldingInsects offer an ideal model system for studying the molecular details of olfaction. Mate-seeking behavior in moths (Insecta, Lepidoptera) is under strong selection pressure, and each moth species responds with exquisite sensitivity to a limited number of fatty acid-derived pheromones. Perception of the female sex pheromone by male moths is mediated by proteins located in the sensory hairs of the antennae (Vogt, 1987;Vogt et al., 1990b;Prestwich, 1992). These include soluble pheromone binding

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Prestwich

1993

Protein Science

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“…The 5Ј primer also contained a ribosomal binding site, a translational spacer element, an N-terminal Met, and the first 5 amino acids of HLA-E exon 2. PCR products were ligated into pHN1ϩ vector (22) and expressed in Escherichia coli strain UBS (23). ␤ 2 -Microglobulin (␤ 2 m) in pHN1ϩ was kindly provided by D. C. Wiley (Harvard University, Cambridge, MA) and expressed in E. coli strain XA90.…”

Section: Methodsmentioning

confidence: 99%

HLA-E Allelic Variants

Strong¹,

Holmes²,

Li³

et al. 2003

Journal of Biological Chemistry

264

114

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Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (E R ) is replaced by a glycine in HLA-E*0103 (E G ). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-E G complexed with two distinct peptides were determined, and both were compared with the HLA-E R structure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.

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“…The 5Ј-lpxQ/1 primer (5Ј-C-GCGCAAGCTTAGGAGGAATTTAAAATGACATATGCGCTGCGTT-CTTCCG-3Ј) was designed for ligation of lpxQ into lac promoter-driven vectors, such as the shuttle vector pRK404a. This primer also incorporates a HindIII site (in italics), a ribosome-binding site (underlined sequence), and a translational spacer element between the ribosomebinding site and the ATG start codon (33). The sequences in bold type correspond to the N-terminal coding region of lpxQ.…”

Section: Quantitative Assay For Measuring the Conversion Of [ 14 C]b mentioning

confidence: 99%

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

Que-Gewirth

Karbarz

Kalb

et al. 2003

Journal of Biological Chemistry

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An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosaminecontaining precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the ϳ20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O 2 for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.As demonstrated in the accompanying article (1), the outer membranes of Rhizobium leguminosarum and Rhizobium etli contain an unusual oxidase that converts the proximal glucosamine unit of 1-dephosphorylated lipid A (or related molecules) to a novel 2-aminogluconate moiety. The membranes of Sinorhizobium meliloti and Escherichia coli do not normally contain such an oxidase activity. Although the function of this unusual covalent modification of lipid A is unknown (2-4), the existence of an oxidative enzyme in the outer membrane of a Gram-negative bacterium is without precedent. The few outer membrane enzymes described to date are all phospholipases (5, 6), acyltransferases (7, 8), or proteases (9). Other characterized outer membrane proteins function either as porins or specialized transporters (10 -12). The presence of the oxidase in outer membranes of certain strains of Rhizobium suggests that lipid A oxidation (1), when it occurs, is a late event in lipopolysaccharide assembly.Given the considerable progress that has recently been made with the structural biology of outer membrane proteins by x-ray crystallography (12) and NMR spectroscopy (8, 13) and the great interest in li...

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Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.

Cited by 99 publications

References 52 publications

Bacterial expression and photoaffinity labeling of a pheromone binding protein

Bacterial expression and photoaffinity labeling of a pheromone binding protein

HLA-E Allelic Variants

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

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