Abstract: The aim of present study was to compare the biological activity of plasmid purified by CsCl2 buoyant density gradient centrifugation and anion exchange HPLC elution from alkaline cell lysates. The recombinant plasmids were purified from E. coli clones by anion exchange HPLC and CsCl2 density gradient centrifugation. Purified samples were digested with restriction endonuclease and the ligated recombinant plasmids were used to transform E. coli HB101 strain. The CsCl2 purified plasmid DNA exhibited higher degree of purity than HPLC purified plasmid. However, transformation efficiency was higher for HPLC purified samples; recombinant pUC8 in VE144 varied between 2x103 to 3.5x103 for crude sample, 2.52x103 to 4.2x103 for CsCl2 density gradient purified sample and 7.5x104 to 1.15x105 transformants/μg of HPLC purified sample. The pBR322 in VE145 also showed transformation efficiency as 3.9x103 to 1.2x104 for crude sample, 5.6x104 to 6.3x105 transformants/μg of CsCl2 purified and 4.5x105 to 5.04x106 transformants/μg of HPLC purified plasmid. Due to the higher transformation efficiency and less time consumption, plasmid purification by HPLC can imply major impact on recombinant DNA technology.
INTRODUCTION
Plasmids are used as a cloning vehicle for recombinant DNA technology. A complete set of plasmid that is covalently closed circular plasmid will be used in further transformation for or particular inserts are used in analytical or preparative mode. For such work one can need plasmid DNA in very high purity. The conventional method of plasmid preparation was precipitation with polyethylene glycol which is tedious or banding them, in a CsCl2 density gradient requires prolonged overnight ultra centrifugation at high speed and addition of ethidium bromide and caesium chloride (CsCl2) has to be removed by organic extraction and dialysis, respectively. All of which might raise concerns about residues in the final product.
Plasmids can be purified by different HPLC methods. Purification by chromatography is considered as the method with highest resolution for producing plasmid DNA proficient for therapeutic applications (Urthaler et al., 2005a). The commonly used purification techniques are anion exchange (Hines et al., 1992; Eon-Duval and Burke, 2004), hydrophobic interaction (Diogo et al., 2000), reverse phase (Weiner et al., 1998) and size-exclusion chromatography (Whisenant et al., 1998). Most methods are based on plasmid isolation by standard procedures (Maniatis et al., 1989) yielding the cleared cell lysate and the resultant plasmid has to be separated by HPLC from the remaining contaminants of host cell origin such as proteins, small RNAs, residual chromosomal DNA and lipids (Schluep and Cooney, 1998).
Purified plasmid molecules play an essential role in gene therapy, genetic vaccination (Stadler et al., 2004) and in molecular biology laboratories to get stringent results. The avoidance of critical reagents such as animal-derived compounds (enzymes), organic solvents and detergents will significantly reduce the ill effects on patient and operator safety (Moreira et al., 2005). The purpose of the present study was to compare the biological activity of plasmid purified by CsCl2 buoyant density gradient centrifugation and anion exchange HPLC elution from alkaline cell lysates.
MATERIALS AND METHODS
The study was conducted at Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, India during January 2006 to June 2006.
Bacterial Strains and Plasmids
The E. coli clones VE 140, VE 141, VE 142, VE 143, VE 144 and VE
145 harboring plasmid pIC20R, pIC20R, pIC19H, pIC19H, pUC8 and pBR322 respectively,
used in this study were obtained from School of Biotechnology, Madurai Kamaraj
University, India. E. coli HB101, sensitive to ampicillin was used for
transformation analysis (Table 1).
Plasmid Isolation from Bacteria
Plasmid DNA molecules were extracted by the alkaline lysis method of Maniatis
et al. (1989). The isolated plasmids were suspended in 3 mL of TE buffer,
pH 8.0. From this processed sample, 2 mL aliquots were taken for CsCl2
density gradient purification and 1 mL was allotted for HPLC analysis in which
100 μL injections were made.
Plasmid Purification by CsCl2-EtBr Density Gradient Centrifugation
All plasmid DNA were isolated by alkaline lysis method followed by caesium
chloride-ethidium bromide density gradient ultracentrifugation by standard procedures
(Maniatis et al., 1989). Two milliliter of plasmid DNA extracted by
alkaline lysis method was purified by isopycnic banding in CsCl2
with 4.4 g CsCl2, 0.25 mL of 10% (w/v) EtBr and made up to 4 mL by
TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) and centrifuged at 50 k for 14.5
h at 20°C. After centrifugation, the banded plasmids were removed and extensively
dialysed against TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) and stored at -20°C.
Purity of plasmids was checked by UV absorbance at A260:A280
and 0.8% agarose gel electrophoresis.
Plasmid Purification by HPLC
The HPLC purification of plasmid DNA was performed by having Shimadzu liquid
pump: LC-6AD, system controller: SCL-6B, UV-Vis detector: (195-700 nm)-SPD-6AV,
data processor: CR-5A, column: DEAE 5PW, mobile phase: buffer A (25 mM Tris-HCl,
1 mM EDTA, pH 8.0) and Buffer B (25 mM Tris-HCl, 1 mM EDTA, pH 8.0, 1 M NaCl),
flow rate: 0.5 mL min-1 and wavelength: 260 nm.
| Table 1: | Details of E. coli clones and recombinant plasmids used |
The 100 μL of crude plasmids were injected and different fractions were collected for all the peaks. The plasmids purified by CsCl2-EtBr density gradient centrifugation were also injected at 10 μL volume and observed the peak with retention time. The fractions corresponding to the first major peak and second major peaks were collected separately and treated with 1/10th volume of 5 M ammonium acetate and 2.5 volume of 95% ethanol for precipitation. Precipitated pellets were washed with 75% ethanol and stored in 50 μL TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0).
Analysis of Plasmid DNA
Concentrations of anion exchange-HPLC, CsCl2-EtBr density gradient
purified plasmids and crude plasmids were determined by taking UV absorbance
readings at 260 nm and purity of the samples were determined by calculating
the ratio of UV absorbance at 260/280 nm. Total yield of plasmids were calculated
by multiplying the concentration by the volume of recovered eluate. The Samples
were run on a 0.8% TBE agarose gel at 60 volts for 1 h, visually inspected under
UV-trans illuminator to confirm their yield and purity and photographed.
Restriction Enzyme Digestion
Restriction enzymes and T4 DNA ligase were obtained from Promega.
The unpurified and purified plasmids pIC20R, pIC20R, pIC19H, pIC19H, pUC8 and
pBR322 were digested with 2 units of BamHI, HindIII, EcoRI,
EcoRI, MluI and PstI respectively for 3 h at 37°C.
After the digestion samples were kept in ice for 2 min, then kept at 65°C
for 10 min and kept in ice. Digested products were checked on 0.8% agarose gel.
Transformation Experiment in E. coli HB101 Strain
The transformation experiments were done as reported by Cohen et al.
(1972) and performed in triplicates. Fifty nanogram of restriction digested
recombinant plasmids were ligated with the respective insert DNA molecule using
T4 DNA ligase, 10 mM ATP at 14°C for 6 h and the ligated recombinant
plasmids (50 ng) were transformed to E. coli HB101 competent cells by
heat shock method and plated on LB plates containing ampicillin (50 μg
mL-1) and incubated at 37°C. The positive and negative controls
were maintained by having 50 and 0 ng of respective plasmid DNA. The colonies
were counted and the transformation efficiencies were calculated as the number
of transformed colonies obtained divided by the amount of plasmid DNA contained
with in the spreading volume on the ampicillin plate.
RESULTS
Purification of Plasmids by HPLC and CsCl2 Buoyant Density Gradient
Centrifugation
The unpurified recombinant plasmids prepared from various strains were passed
through DEAE-5PW column. The elution pattern monitored by absorption at 260
nm is shown in Fig. 1. The concentration and purity were analyzed
by recording absorbance at 260 and 280 nm. Table 2 summarizes
the plasmid concentration in different purification strategies and their recovery
percentage.
Purity of the CsCl2 density gradient purified recombinant plasmids
were checked by anion exchange HPLC. The HPLC results showed that the plasmid
pIC20R from VE140 clone have two peaks of retention time 6.202 and 27.248 min
and CsCl2 density gradient purified sample showed the peaks of 6.36
and 26.208 min. In pIC20R of VE141 injection, two peaks were observed (6.314
and 26.027 min) and CsCl2 density gradient purified samples showed
two peaks of 6.342 and 26.595 min. The pIC20H from VE142 showed two peaks (6.364
and 25.647 min) and its CsCl2 density gradient purified counterparts
showed two peaks of 5.9 and 26.487 min. In general all the clones exhibited
similar kind of chromatogram i.e., first peak between 5.9 to 6.828 and second
peak between 25.647 to 27.665 min and the fraction a, which corresponds to first
peak is characterized by having maximum amount of protein, RNA and other contaminants.
| Fig. 1: | Elution profile of recombinant plasmids from an anion-exchange HPLC column. 1, 2, 3, 4, 5 and 6 represents the recombinant plasmids from clones VE140, VE141, VE142, VE143, VE144 and VE145, respectively. (1a-3b) depicts the CsCl2 density gradient purified samples and (4a-6b) represents the elution pattern of plasmid samples. The first peak eluted near the void volume denotes the contaminants and second peak denotes the elution of plasmids |
| Table 2: | Concentration and purity of plasmids by UV absorbance at 260/280 |
| Fig. 2a and b: | Restriction pattern of purified plasmids Lane M-λ HindIII size marker; 1, 2 and 3 represents HPLC, CsCl2 purified and crude plasmids, (a) denotes undigested samples; (b) represents digested products of plasmids |
The fraction b of second peak represents the pure form of plasmid DNA and agarose gel electrophoresis of fraction b showed the presence of concatemeric, linear, nicked and supercoiled form of plasmid DNA.
Restriction Digestion Assay
All the recombinant plasmids were digested with respective restriction endonuclease
as shown in Table 1. Agarose gel electrophoresis of digested
samples showed the complete excision of insert DNA from plasmid backbone and
there were no difference in the pattern of bands in the 0.8% agarose gel for
all the crude, HPLC and CsCl2 density gradient purified plasmid samples
(Fig. 2a and b).
Transformation Assay
The transformation experiments were carried out for the two clones namely
VE144 and VE145. Transformation efficiency of clone VE144 varied between 2x103
to 3.5x103 for crude sample, 2.52x103 to 4.2x103
for CsCl2 density gradient purified sample and 7.5x104
to 1.15x105 transformants/μg of HPLC purified sample. The VE145
also showed similar transformation efficiency (i.e.,) 3.9x103 to
1.2x104 for crude sample, 5.6x104 to 6.3x105 transformants/μg
of CsCl2 buoyant density gradient purified plasmids and 4.5x105
to 5.04x106 transformants/μg of HPLC purified samples. The presence
of plasmids in the Ampr colonies of transformation assays were carried
out and all the Ampr colonies showed the presence of respective recombinant
plasmids.
DISCUSSION
The traditional and newly developed DNA purification procedures have the goal to remove the toxic components and other cell debris from the cell extracts. A number of commercial kit-based DNA purification techniques have become popular but the large scale cost effective purification of DNA remains a technological challenge and make the DNA purification as the expensive one. Plasmid purification by anion exchange HPLC resulted in highly efficient plasmid molecules free from general contaminants of chromosomal DNA, RNA and proteins. The polynucleotides are negatively charged and they are independent of the buffer conditions in the anion-exchange chromatography (Urthaler et al., 2005b) and it may be the prime factor in yielding the biologically efficient and highly purified plasmid DNA by HPLC equipped with DEAE column. The HPLC purified plasmid DNA pools were devoid of residual contaminants and the percentage of recovery was in the range of 75.29 to 83.76. When compared to CsCl2 buoyant density gradient centrifugation the plasmid purification by HPLC is a less tedious process and it can be employed both small and large scale purification of plasmid DNA molecules.
Transformation efficiency of HPLC purified samples was higher when compared to CsCl2 buoyant density gradient purified plasmid DNA and both the preparations showed more or less similar transformation efficiency. However, the larger plasmids can be easily lost during CsCl2 density gradient centrifugation (Corneau et al., 2001) but the HPLC system can separate the nucleic acids in order of size with the smaller fragments eluting first and can provide an effective separation of fragments as small as 10 bp to 50 kbp by ion exchange column and HPLC purification takes lesser time (about 30 min for separation) than CsCl2 density gradient centrifugation. From the above results we can conclude that plasmid purification by anion exchange HPLC is best performed in transformation experiments and can be used for genetic engineering and other purposes.
ACKNOWLEDGMENTS
We thank Prof. K. Veluthambi, School of Biotechnology for providing E. coli clones for this study and we sincerely thank International Atomic Energy Agency, Vienna, Austria for the financial support.