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Biomedical subjects

D K Summers

Publications and source records attributed to D K Summers.

17 recordsLinked to original sources
PubMed2005

Recombinant protein secretion in Escherichia coli.

The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.

Bacterial Proteins↗
PubMed2004

Studies of single-chain antibody expression in quiescent Escherichia coli.

Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from lambdaP(L) under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from lambdaP(R). Shifting the culture from 30 to 42 degrees C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD(600)] of 3.5) was 37 mg x liter(-1), compared to a maximum of 13 mg x liter(-1) in the control culture (final OD(600) of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD(600) of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg x liter(-1). A control culture with a similar feed reached an OD(600) of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg x liter(-1). Quiescent cells at an OD(600) of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg x liter(-1) was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.

Biotechnology↗
PubMed2001

Monomer-dimer control of the ColE1 P(cer) promoter.

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Aminopeptidases↗
PubMed1999

The quiescent-cell expression system for protein synthesis in Escherichia coli.

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.

Bacterial Proteins↗
PubMed1998

Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis.

Dimers of plasmid ColE1 are converted to monomers by site-specific recombination, a process that requires 240 bp of DNA (cer) and four host-encoded proteins (XerC, XerD, ArgR and PepA). Here, we propose structures for nucleoprotein complexes involved in cer-Xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and DNA curvature. We propose that, in the nucleoprotein complex at a single cer site, a PepA hexamer acts as an adaptor, connecting the heterodimeric recombinase (XerCD) to an ArgR hexamer. This provides a protein core around which the cer site wraps, its exact path being defined by strong sequence-specific interactions with ArgR and XerCD, weak interactions with PepA and sequence-dependent flexibility of cer. The initial association of single-site complexes (pairing) is proposed to occur via an ArgR-PepA interaction. Pairing between sites in a plasmid dimer is stabilized by DNA supercoiling and is followed by a structural isomerization to form a recombination-proficient synaptic complex. We propose that paired structures formed between sites in trans are too short-lived to permit synaptic complex formation. There is thus an energetic barrier to inappropriate recombination reactions. Our proposals are consistent with a wide range of experimental observations.

Acetyltransferases↗
PubMed1994

The origins and consequences of genetic instability in prokaryotes.

The causes of genetic change include errors in replication and repair, transposition and recombination. Together, these processes generate a reservoir of genetic variants from which selection will amplify any changes which increase host fitness. Recombination, transposition and slippage during replication may all result in large-scale DNA rearrangements. Homologous and illegitimate recombination generate deletions, inversions, duplications and fusions. Intramolecular transposition can cause deletion or inversion of DNA sequences adjacent to the mobile element, while intermolecular movement may lead to fusion of donor and target replicons when transposition is replicative. Of crucial importance is how genetic variation affects the fitness of the host cell. For a neutral variant the probability of eventual fixation is small, but changes which increase host cell fitness pose a more serious problem because these cells will eventually dominate the culture. The most effective way to combat the proliferation of variant plasmid cloning vectors is to block the processes by which they arise. Cloning vectors should be "stress-tested" and DNA sequence analysis programs can be used to screen for sequence repetition which should be removed, as far as possible, from both vectors and cloned sequences. It is also important that vectors place modest metabolic demands on their hosts in order to minimise the potential increase in fitness associated with changes in plasmid structure.

Biotechnology↗
PubMed1993

ColE1 multimer formation triggers inhibition of Escherichia coli cell division.

Multimer formation and consequent copy number depression are acknowledged causes of multicopy plasmid instability. Multimer resolution sites (among which ColE1 cer is best-characterized) have been identified in a variety of plasmids. They participate in the conversion of multimers to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. We show that multimer resolution alone is insufficient to ensure stable maintenance of ColE1-like plasmids in a recombination-proficient host. The expression of Rcd, a transcript encoded within cer and expressed in multimer-containing cells, is also required. The appearance of Rcd correlates with the inhibition of division of multimer-containing cells, presumably allowing time for the conversion of multimers to monomers by site-specific recombination.

Bacterial Proteins↗
PubMed1993

Multicopy plasmid instability: the dimer catastrophe hypothesis.

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains. Most cells in the population contain only monomers; dimers are confined to a small subpopulation from which plasmid-free daughters arise at high frequency. In a rec+ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.

Cell Division↗
PubMed1991

The kinetics of plasmid loss.

Instability of bacterial cloning vectors can present a serious problem when direct selection for plasmid-encoded phenotypes is undesirable, ineffective or impractical. Antibiotic selection may provide a satisfactory solution in enclosed fermentors but not where recombinant organisms are part of complex microbial consortia after release into the outside environment. In the past decade there has been significant progress towards understanding the causes of plasmid loss and the lessons learned from these studies can be used in the design of a new generation of stable vectors.

Kinetics↗
PubMed1989

Derivatives of ColE1 cer show altered topological specificity in site-specific recombination.

ColE1 contains a 250-bp sequence (cer) which is required in cis for the conversion of plasmid dimers to monomers. Recombination between cer and parB (a dimer resolution site from plasmid CloDF13) occurs in vivo at low frequency. The properties of the resulting hybrid sites have been studied. The type I hybrid closely resembles wild-type cer. It supports intramolecular recombination and requires the products of the chromosomal xerA, xerB and xerC genes together with the 250-bp site. In contrast, the type II hybrid (although differing from type I by only 2 bp) functions independently of the topological relationship of the participating sites, supporting both inter- and intramolecular recombination. Furthermore, recombination between type II sites is independent of the products of the xerA and xerB genes and requires a site of less than 50 bp.

Bacteriocin Plasmids↗
PubMed1988

Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site.

Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.

Chromosome Deletion↗
PubMed1984

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability.

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.

Ampicillin↗
PubMed1983

Plasmid replication in a temperature-sensitive chromosome replication mutant of Staphylococcus aureus.

Replication of the antibiotic resistance plasmids pI258, pT10501 and pC221 has been investigated in a mutant of Staphylococcus aureus NCTC 8325, which is temperature-sensitive for the initiation of chromosome replication. Replication of pI258 stopped rapidly at the nonpermissive temperature, whilst replication of pT10501 and pC221 continued (although at a lower rate than in the wild-type). It is proposed that the product of the mutant gene may be required directly for pI258 replication, but not for replication of pT10501 or pC221.

Chromosomes, Bacterial↗
PubMed1982

Characterization of a temperature-sensitive DNA replication mutant of Staphylococcus aureus.

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive-temperature (42 degrees C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 degrees C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 degrees C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 degrees C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 degrees C.

Chloramphenicol↗