Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography.

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h. The process delivers sc MCs that are virtually free from MP, PP, RNA, and protein impurities.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.

Development of a nicking endonuclease-assisted method for the purification of minicircles.

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography (HIC) step. At the early stage of vector design, a site for the nicking endonuclease Nb.BbvCI was strategically placed in the MP part of the PP backbone. A process was then established that involves E. coli culture and recombination of PPs into target MC, cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. Next, an in vitro digestion step was carried out with Nb.BbvCI to nick of one of the strands of the MPs and of non-recombined PPs by Nb.BbvCI. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular (oc) forms whereas sc MCs remain unaffected. Finally, sc MC was isolated from oc DNA molecules (oc MPs, oc MC) and RNA by performing HIC with a phenyl-Sepharose column using a series of elution steps with decreasing ammonium sulfate concentrations. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.

Towards effective non-viral gene delivery vector.

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.

Induction and carbon catabolite repression of phenol degradation genes in Rhodococcus erythropolis and Rhodococcus jostii.

Rhodococcus erythropolis CCM2595 is able to efficiently utilize phenol and other aromatic compounds. We cloned and sequenced its complete gene cluster - catA, catB, catC, catR, pheR, pheA2, pheA1 - involved in the ortho-cleavage pathway of phenol. The activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, was found to be induced by phenol. When both phenol and succinate were present in the medium, phenol hydroxylase activity decreased substantially. To analyze the regulation of phenol degradation at the transcriptional level, the transcriptional fusions of the divergently oriented promoters PpheA2 and PpheR with the gfpuv reporter gene were constructed. The promoters driving expression of the genes of the pheR-pheA2pheA1 cluster were localized by determining the respective transcriptional start points. Measurements of GFP fluorescence as well as quantitative RT-PCR revealed that expression of the phe genes is induced by phenol at the transcriptional level. The transcription of pheA2A1 and pheR was repressed by succinate, whereas no repression by glucose or glycerol was observed. Activation of the R. erythropolis CCM2595 pheA2 promoter by PheR, an AraC-type transcriptional regulator, was demonstrated by overexpression of the pheR gene. Analysis of the transcriptional regulation of two similar phe clusters from R. jostii RHA1 by various substrates showed that the type of carbon catabolite repression and the temporal transcriptional pattern during cultivation are different in each of the three phe clusters analyzed.

On the dual effect of glucose during production of pBAD/AraC-based minicircles.

Minicircles are promising vectors for DNA vaccination, gene or cell therapies due to their increased transfection efficacy and transgene expression. The in vivo production of these novel vectors involves the arabinose inducible excision of a parental molecule into a minicircircle and a miniplasmid bacterial backbone. Tight control of recombination is crucial to maximize minicircle yields and purity. In this work, a minicircle production system was constructed that relies on the enzymatic activity of ParA resolvase, a recombinase that is expressed under the transcription control of the arabinose inducible expression system pBAD/AraC, and on Escherichia coli BWAA, a strain improved for arabinose uptake. Undesired recombination already after 4h of incubation in Luria-Bertani broth at 37 °C was observed due to the leaky expression from pBAD/AraC. While addition of glucose to the growth media repressed this leaky expression, it triggered a pH drop to 4.5 during exponential phase in shake flasks, which suppressed growth and plasmid production. The quantitative PCR analysis confirmed only few copies of high-copy number plasmid inside of the E. coli cells. To ensure the stability of minicircle-producing system, seed cultures should be grown at 30 °C with glucose overnight whereas cells for minicircle production should be grown in shake flasks at 37 °C without glucose up to early stationary phase when the recombination is induced by addition of arabinose.

Isolation of a lipase-secreting yeast for enzyme production in a pilot-plant scale batch fermentation.

The production of lipase by twenty-nine yeasts isolated from the phylloplane of Hibiscus rosa-sinensis was evaluated. The highest lipase producers were Pseudozyma hubeiensis HB85A, Debaryomyces occidentalis-like HB83 and Cryptococcus sp. HB80. P. hubeiensis HB85A batch fermentations were carried out in a bioreactor and lipase production improved 3.2-fold as compared to flask submerged cultures. The production process was significantly reduced from 48 h (in flasks) to 18 h (in the bioreactor). The better hydrolytic activity was achieved with C16 p-nitrophenyl ester. Maximal activity was observed at pH 7.0, the optimum temperature was 50 degrees C at pH 7.0 and the enzyme was stable at 30 and 40 degrees C. The lipolytic activity was stimulated by Mg(2+), K(+) and Ba(2+) salts and EDTA and slightly inhibited by Ca(2+) salts. Non-ionic detergents such as Triton X-100, Tween 80 and Tween 20 strongly stimulated lipase activity, whereas SDS inhibited it. The lipase was stable in iso-octane and hexane at 80%.