Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli

Elvin, Christopher M.; Thompson, Phillip R.; Argall, M. E.; Hendry, Philip; Stamford, N. Patrick J.; Lilley, Penelope E.; Dixon, Nicholas E.

doi:10.1016/0378-1119(90)90503-j

Cited by 123 publications

(79 citation statements)

References 10 publications

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“…DNA polymerase III subunits ␣ and ␤ were purified by modifications of published procedures using strains containing wild-type dnaE ϩ and dnaN ϩ derivatives of the -promoter vector pCE30 (12). The ␦ subunit was prepared by using strain BL21(DE3)͞pLysS͞pET␦, essentially as described (13).…”

Section: Methodsmentioning

confidence: 99%

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple

Kongsuwan

Wijffels

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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309

368

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The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. T he replication of DNA in eubacteria involves many proteins organized into a complex multifunctional machine termed the replisome. A central enzyme is the multisubunit DNA polymerase III holoenzyme. In Escherichia coli, and probably in most other eubacteria, the DnaE ortholog (␣ subunit) is in the core of the replicative polymerase, whereas in many Grampositive organisms a related enzyme, PolC, is proposed to have this function (1). The processivity of the polymerase is conferred by the direct interaction of the ␤ subunit (clamp protein) of DNA polymerase III (2, 3), with the DnaE (and presumably PolC) subunits. ␤ is loaded onto DNA by a clamp loader comprised of single ␦ and ␦Ј subunits and four ͞␥ subunits (1). The ␤ dimer thence encircles the DNA without actually binding to it. In addition to DnaE, three other E. coli DNA polymerases appear to interact with ␤. PolB (DNA polymerase II) is involved in DNA repair (4) and the addition of ␤ and the clamp loader increases its processivity in vitro (5, 6). Similarly, ␤ and the clamp loader together increase both the processivity (7) and efficiency (8) of DNA synthesis by DNA polymerase IV (DinB). ␤ also appears to play a similar role in the activity of DNA polymerase V (8) (the UmuDЈ 2 UmuC complex) and the UmuD subunit has been shown to bind to ␤ (9).Experimental evidence shows that at least some ␤-binding proteins can interact productively with ␤ from heterologous species. For example, PolC subunits from Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1, 10, 11). In contrast, E. coli DnaE cannot use ␤ from the other species (11), the E. coli clamp loader complex cannot load S. aureus ␤ (11), and the S. pyogenes clamp loader complex cannot load E. coli ␤ (1).In the absence of any experimentally identified ␤-binding sites in proteins, a bioinformatics approach was undertaken to identify putative ␤-binding motifs. The role of the putative motif was then examined by yeast two-hybrid and peptide-binding experiments with native and modified sequences. Materials and MethodsSources of Amino Acid Sequences. Amino acid sequences and alignments were derived from: PSI-BLAST of the protein database at the National Center for Biotechnology Information (NCBI), and BLAST of preliminary sequence data from NCBI at http:͞͞ www.ncbi.nlm.nih.gov͞Microb_blast͞unfinishedgenome.html, Institute for Genomic Research at http:͞͞www.tigr.org, Department of Energy Joint Genome Institute at http:͞͞spider.jgipsf.org͞JGI_microbial͞html͞, Sanger Center at http:͞͞ www.sanger.ac.uk͞DataSearch͞omniblast.shtml, and ERGO at http:͞͞wit.integratedgenomics.com͞IGwit͞. Alignments of available sequences of all members of eubacterial protein families known to bind to ␤ were compiled with manual editing in regions of variab...

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Section: Methodsmentioning

confidence: 99%

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple

Kongsuwan

Wijffels

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

309

368

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“…Protein Purification-Overproduction of DnaG, DnaG-(1-433), and DnaG-C was achieved in E. coli strains containing plasmid derivatives of vector pCE30, pMA200U (28), or pND706 (29), respectively, with appropriately constructed genes under control of thermoinducible tandem bacteriophage p R and p L promoters. Construction of pPL195 (dnaG ϩ ) (21) and pKL1176 (dnaG-C ϩ ) (30) have been described.…”

Section: Methodsmentioning

confidence: 99%

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Oakley

Loscha

Schaeffer

et al. 2005

Journal of Biological Chemistry

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107

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During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB 6 . Determination of the 2.8-Å crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primasehelicase complex at replication forks.

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“…Briefly, SSB was overproduced in E. coli strain BL21(λDE3)recA [49] containing plasmid pND73 [50], with the ssb gene under the control of thermoinducible bacteriophage λ p R and p L promoters. Uniformly 15 N-labeled SSB was overproduced in M9 minimal media containing 15 NH 4 Cl (Cambridge Isotope Laboratories, Andover, MA) as the sole source of nitrogen.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason

Jergic

et al. 2013

J. Am. Soc. Mass Spectrom.

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Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions AbstractSingle-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry. Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNAbinding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange.Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

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Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli

Cited by 123 publications

References 10 publications

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

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