Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis

Production of recombinant proteins often interferes with the physiology of the host organism by causing stress responses. In recombinant Escherichia coli, the cellular content of ColE1-derived plasmids and, consequently, the synthesis of the constitutively synthesized plasmid-encoded proteins generally increases after a temperature upshift. Simultaneous induction of inducible recombinant proteins that are synthesized at high levels and tend to form inclusion bodies, however, attenuates the plasmid amplification. This phenomenon was observed using temperature- as well as IPTG-inducible expression systems. Thus, high-level recombinant gene expression in connection with inclusion body formation does not only interfere with host cell but also with plasmid-related functions.     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Isolation of Plasmid DNA rescued from single colonies of<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> by means of rolling circle amplification

We report a simple method to isolate plasmids from single colonies ofAgrobacterium tumefaciens by means of rolling circle amplification. The amplified DNA can be digested by restriction enzymes for plasmid verification and transformed intoEscherichia coli for plasmid rescue. Compared with conventional procedures, this method eliminates liquid culturing ofAgrobacterium cells and subsequent DNA isolation and enables large-scale plasmid analyses.     

Plasmid distribution in Escherichia coli urinary isolates with special reference to aerobactin and colicin production

Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified cases of urinary tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed an association of aerobactin and colicin production with the distribution of plasmid bands having a median value of 2.33 and 1.72 (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For colicin producers, the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains.     

Plasmid macro-evolution: selection of deletions during adaptation in a nutrient-limited environment

A study was made of the intra-and inter-population variability of the main traits involved in Trichogramma (T. brassicae and T. cacoeciae) efficiency in host exploitation: longevity, fecundity, progeny viability, progeny sex ratio and progeny allocation. The analysis of isofemale strains shows that differences in progeny viability, progeny sex ratio and progeny allocation are transmissible and relatively stable over two successive generations. Comparison of three strains of T. brassicae originating from different locations, demonstrates differences in fecundity, progeny sex ratio and progeny allocation. Differences in host exploitation strategies also exist between two sympatric populations of T. brassicae and T. cacoeciae. No significant correlation appears between the traits which discriminate populations. The ecological and evolutionary significance and the agronomical importance of the results are discussed.     

Improved stability and expression of a recombinant shuttle plasmid in Escherichia coli during fedbatch cultivation

The effect of higher cell densities on the expression and segregational stability of a recombinant E. coli- B. subtilisshuttle plasmid coding for carboxymethylcellulase (CMCase) activity, was studied in E. coli DH5. Of the various feeding policies adopted for maximal expression and stability, exponential feeding resulted in the highest biomass of 15g dry cell weight (DCW) l^–1 and plasmid stability of 45%. A CMCase activity of 11400 Uml^–1 was achieved as compared to 230 Uml^–1 during batch cultivation. In the case of other feeding strategies viz., constant feeding, linear feeding or intermittent feeding, the plasmid stability varied between 20% to 60%. Biomass achieved ranged from 5.0 g DCW l^–1 to 9.0 g DCW l^–1 and enzyme activities were between 2550 Uml^–1 and 6000 Uml^–1.     

Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin

A significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, Paraoxon, to non-toxic materials by co-expression of organophosphorus hydrolase (OPH) under trc promoter and Vitreoscilla hemoglobin (VHb) under O₂dependent nar promoter. VHb-expressing whole cells had significant enhancement of OPH activity (48%, 18.7 vs. 27.8 unit l^–1) and bioconversion efficiency V _max/K _m (44%, 0.14 vs. 0.2 min^–1) compared to VHb-free system.     

A simple procedure to obtain plasmids suitable for transfecting mammalian cell lines

A simple procedure to obtain plasmid preparations, suitable for transfecting mammalian cell lines using a calcium phosphate co-precipitation technique, is described. The protocol is based on the purification of plasmid DNA by double gel-filtration chromatography on Sephacryl S-1000 and additional slight modifications to the original transfection procedure. The purity of plasmid preparation was verified by analytical methods. The resulting preparation efficiently transfected NIH-3T3 cells.The authors are with the National Center for Scientific Research, Molecular Biology Department, Biotechnology Branch, POB 6880, Havana, Cuba.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli

A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

The influence of local plant growth conditions on non-fastidious bacterial contamination of meristem-tips ofHydrangea culturedin vitro

A high incidence ofin vitro bacterial contamination (69%) has been detected in meristem-tip explants ofHydrangea from widely differing locations in Ireland and the UK. The bacteria were characterised by API 20E biochemical test kits and by fatty acid profile analysis. The results obtained from the different methods were compatible and anomalies were explicable in terms of the limitations of the respective methods. The majority of the isolates were environmental or animal-associated bacteria with clusters ofEnterobacter isolates in Dublin, and ofEscherichia coli in the main Cork location. A cluster of Pseudomonads was detected in the Derby (UK) plants. The main association was between the location and the contaminant clusters. The main finding was that the nature of organic soil amendments may influence inoculum for the contamination of plants and the conclusion was that fertilisation with organic materials should be avoided in the preparation of plants for micropropagation.     

Coexistence of Genetically Engineered Escherichia coliStrains and Natural Microorganisms in Experimental Aquatic Microcosms

In experimental aquatic microcosms (AMCs), the population of the Escherichia colistrain Z905 harboring the recombinant plasmid pPHL7 (Ap^rLux⁺) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adaptedE. colicells could coexist with the native AMC microflora for one year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coliZ905-33 (pPHL7) cells were more competitive than the nonadapted cells.     

Expression and preparation of recombinant hepcidin in Escherichia coli

Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.     

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml^–1) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml^–1 dropped to 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml^–1 probably reflects a loss of cell division capability rather than cell death.     

A high cell density fermentation strategy to produce recombinant malarial antigen in <Emphasis Type="Italic">E. coli</Emphasis>

A high cell density cultivation method was developed to produce recombinant PvRII, a malaria vaccine candidate, in E. coli for use in vaccine studies. Cells were grown in completely defined media and glucose was fed to achieve a specific growth rate of 0.12 h^–1 until cells reached 55 g dry wt l^–1. Culture was then induced with 1 mm IPTG and cells were further grown for 4 h to reach 85 g dry wt l^–1 at 0.1 h^–1. Recombinant PvRII was purified from inclusion bodies under denaturing conditions using metal affinity chromatography which yielded 10 mg PvRII g^–1 dry wt. After refolding, PvRII was greater than 98% pure, homogeneous and functionally active in that it specifically bound Duffy positive human red cells.Revisions requested 21 September 2004; Revisions received 29 October 2004