Uronic acid assay
We modified a highly sensitive carbazole analysis assay that specifically quantifies the amount of uronic acid contained in polysaccharides.21
Briefly, 40 µg of each plasmid DNA was diluted in sterile water for irrigation USP (sterile water; B.Braun Medical, Irvine, CA, USA). The standard, for example, glucuronic acid, was diluted from a 1 mg/ml stock solution in sterile water to concentrations ranging from 0.005–0.5 mg/ml, and water as control was also prepared. All samples and controls were placed into 13 × 100 mm glass test tubes. A 0.025 m
sodium tetraborate-decahydrate solution was prepared in concentrated sulfuric acid (specific gravity, 1.84). The borate was added to the sulfuric acid slowly to prevent clumps of fused borax from foaming. A 0.125% carbazole solution was also prepared by dissolving carbozole in absolute EtOH. Just before heating, 3 ml of the borate/sulfuric acid solution followed by 100 µl of the carbazole reagent were added to each tube. Glass disposable pipettes were used to dispense the borate/ sulfuric acid solution. Each tube was vortexed well, covered with a glass marble and placed into a suitable wire rack in a water bath. The water bath was set to boil, and the tubes were boiled for 10 min while maintaining constant boiling. The tubes were then placed at room temperature (RT) for 10 min. Each tube was vortexed and placed at RT for 5 min to allow any air bubbles to dissipate. The samples and the standards were measured for absorbance at 530 nm. All samples and standards were assayed in duplicate. The concentrations of polysaccharides in the samples were calculated on the basis of the linear regression data generated from the glucuronic acid standard curve.
We modified an assay for measuring sub-nanomole amounts of fucose using enzymatic cycling.22
Each test sample was first prepared for analysis by lyophilizing 450 µg of plasmid DNA in a glass vial. In total, 200 µl of 5.5 m
trifluoroacetic acid was added to each DNA sample, and the vials were tightly sealed with Teflonlined caps. The samples were then hydrolyzed by heating for 4 h at 100 °C. The samples were cooled to RT and the trifluoroacetic acid was removed with a passage of stream of argon gas under the fume hood. The DNA residue in each vial was dissolved in 200 µl of sterile water. Fucose standards were prepared ranging from 0.005–1 mg fucose/ml sterile water. Sterile water was used as a standard for the negative control. 200 µl of each test sample and the standards were aliquoted into sterile 1.5 ml eppendorf tubes and placed on ice. Then 50 µl of 1 mg/ml fucose dehydrogenase (Kikkoman Corporation, Walworth, WI, USA) were added to each tube followed by the addition of 50 µl of 200 µm
nicotinamide adenine dinucleotide. The tubes were flicked to mix and incubated at 4 °C for 3 h. Then 50 µl of 1N NaOH were added to each tube. The tubes were flicked to mix and incubated for 10 min at 60 °C to stop the enzymatic reaction. The tubes were cooled to RT and neutralized with 50 µl of 1 m
HCl. In total, 50 µl from each tube were removed and placed into a new sterile 1.5 ml eppendorf tube. Then 250 µl of the cycling reagent was added to each tube that was then flicked to mix. The cycling reagent consists of 200 mm
Tris, pH 8.4, 50 mm
ammonium acetate, 0.5 mm
adenosine di-phosphate, 100 mm
lactate, 5 mm
alpha-ketoglutarate, 20 units/ml lactate dehydrogenase and 20 units/ml glutamate dehydrogenase. The tubes were incubated at RT for 1 h. The reaction was stopped by heating the tubes for 2 min in boiling water. The tubes were chilled on ice and 250 µl of the pyruvate reagent were added to each tube and flicked to mix. The pyruvate reagent consists of 800 mm
imidazole buffer, pH 6.2, 0.45 mm
NADH and 0.06 units/ml lactate dehydrogenase. The tubes were warmed to RT in a water bath for 2 min and then placed at 30 °C in an incubator for 20 min. The reaction was halted by addition of 200 µl of 1.5M HCl. The contents of each tube were transferred to 15 ml Falcon tubes. Then 2.5 ml of 6N NaOH were added to each tube and flicked to mix. The tubes were incubated at 60 °C for 10 min. The tubes were then cooled to RT and 4 ml of sterile water was added to each tube and inverted to mix. In total, 300 µl from each tube were placed in a white-bottomed 96-well microtiter plate (Thermo Labsystems, Thermo Scientific) for fluorescence measurements. Samples were assayed in triplicate on a fluorescence P-E plate reader (FLUOstar OPTIMA, BMG Labtech, Cary, NC, USA) set at the following parameters: excitation filter at 360 nm, emission filter at 465 nm, gain at optimal, lag time at 0 µs, integration time at 40 µsec, flashes at 10 µs and time between move and flash at 0 µs. The concentrations of fucose in the DNA samples were calculated on the basis of the linear regression data generated from the fucose standard curve.
This assay was used to detect lower MWpolysaccharides in plasmid DNA preparations. DTAF was purchased from Molecular Probes (Invitrogen) for use in labeling of polysaccharides in plasmid DNA. For each sample, 80 µg of DNA in 40 µl were transferred into 500 µl eppendorf tubes. One tube containing 40 µl of sterile water was included as a control that was treated identically to the DNA samples. The DNA was precipitated with 10 µl of 3 m NaOAc, pH 5.2, and 200 µl cold 100% EtOH. Each sample was vortexed for 5–10 s. The tubes were incubated for 30 min at −20 °C followed by centrifugation at 10 000 r.p.m. for 4 min at 4 °C. The supernatants were discarded, and 10 µl of 3 m NaOAc pH 5.2 and 200 µl cold 100% EtOH were added to the pellets. The tubes were vortexed and centrifuged as stated above. The DNA precipitation and wash process was repeated, except that only 200 µl cold 100% EtOH was added. The tubes were centrifuged and the supernatant discarded. The pellets were dissolved in 50 µl of 0.1 m Na2CO3, pH 10.5, at RT. A 60 mg/ml slurry of DTAF in 0.1 m Na2CO3, pH 10.5, was prepared in a 1.5 ml eppendorf tube that provided enough slurry to add a total of 15 µl to each tube. The DTAF slurry was kept cold on ice and in the dark in a covered ice bucket. 5 µl of the freshly vortexed DTAF slurry was added to each tube, and the DTAF slurry was vortexed every time before addition to each tube. Sample reactions were kept at RT and in the dark in a covered storage box during the course of the reaction. The addition of 5 µl DTAF to each tube was repeated two more times at 45 and 90 min after the initial addition. After a total of 2.5 h (3 × 45 min beyond each addition of DTAF slurry), the reactions were stopped by the addition of 10 µl of 3 M NaOAc, pH 5.2, and 325 µl cold 100% EtOH. All tubes were placed at −20 °C for 45 min. The tubes were centrifuged at 10 000 r.p.m. for 4 min at 4 °C. The pellets were washed with 25 µl of 3 m NaOAc, pH 5.2, and 500 µl cold 100% EtOH, vortexed and centrifuged as stated above. This process was repeated for a total of three washes. The final wash was performed with 500 µl of cold 100% EtOH, vortexed well and centrifuged as stated above. All of the supernatant was removed from each tube and the pellets were air dried for 20 min in the dark (a longer time would make the pellets difficult to dissolve). The pellets were dissolved in 40 µl of 1 × TAE buffer. For gel analysis a 1% agarose gel was prepared in 1 × TAE buffer without including EtBr. 2 µl of a clear gel loading solution (lacking bromophenol blue or any other dye) and 3 µl of 1 × TAE buffer were added to 5 µl of each DTAF-labeled sample. Bromophenol blue was not added to the DTAF-labeled lanes because it quenches the fluorescence. The DNAwas analyzed on the same gel by adding 5 µl of a 1:100 EtBr solution to 5 µg of DNA and 2 µl of gel-loading solution containing bromophenol blue. About 3 µg of a mix of the high MW DNA marker, lambda HindIII and low MW markers was included and was prepared identically to the other DNA samples containing EtBr. The samples were pipetted into the wells leaving an empty well between each DTAF-labeled sample. All lanes were heavily loaded with 10 µg of plasmid DNA per lane to show that polysaccharides migrate differently from plasmid DNA from identical samples. The 1% TAE-agarose gel was electrophoresed for approximately 3 h at 180 V in 1 × TAE running buffer that did not contain EtBr. The gel was checked periodically with a handheld UV lamp to monitor the progress. The bromophenol blue (MW = 670.0) in the DNA lanes was used to determine the progress of the separation by electrophoresis, and it has a higher MW than unbound DTAF (MW = 495.3). The data () show that all the DNA samples contain polysaccharides that are also found in LPS (lane 10) and in detoxified LPS (D-LPS, lane 8). Furthermore, D-LPS is LPS from which lipid A has been removed. Therefore, removal of lipid A and endotoxin, does not remove the majority of polysaccharides. The only polysaccharides that cannot be visualized on the gel are those that are extremely large MW or highly branched and cannot migrate into the gel pores when subjected to electrophoresis, including the long-chained, branched and high MW CA.
Preparation of CA
We produced CA as previously described13
from the growth medium of SC12078 bacteria K-12, a CA-overproducing strain.20
Briefly, SC12078 was inoculated into 2 l of Luria broth (LB) medium containing 0.4% glycerol and 10 µg/ml chloramphenicol. Use of chloramphenicol is required to maintain this CA-overproducing strain. The culture grew at 37 °C in a shaker incubator at 230 r.p.m. overnight (O/N) to an OD600
between 4.5–4.7. The cells were shaken manually in the flask briefly to obtain more CA into the culture medium. The culture was centrifuged at 6000 × g for 15 min at 4 °C. The supernatants were pooled and the pellet was discarded. The volume of the supernatant was reduced using an Amicon filter apparatus and the YM30 membrane according to manufacturer’s instructions (Millipore, Billerica, MA, USA). Three volumes of icecold EtOH were added to the retentate and placed on ice for 15 min to precipitate the CA. The precipitate was collected by centrifugation at 10 000 × g for 15 min at 0 °C to yield a clear supernatant. The pellet was dissolved in sterile water using the least amount of water required. The mixture was dialyzed in sterile water O/N at 4 °C with three changes. The empty tube that would store the pellet was weighed. The mixture was then placed into this tube and lyophilized to dryness. The tube containing the pellet was then weighed. The final weight of the pellet was determined by subtracting the weight of the empty tube plus cap from the weight of the tube and cap containing the pellet. A 2% solution of the pellet was prepared by dissolving in sterile water. Solid ammonium sulfate was added to saturate the sample to 90%. At this point in the process, both antigen O and CAwere precipitated. The precipitate was collected by centrifugation at 10 000 × g for 15 min at 0 °C to yield a clear supernatant. The pellet was dissolved in sterile water using the least amount of water required. The sample was dialyzed in sterile water O/N at 4 °C with three changes. The sample was lyophilized to dryness and dissolved in 150 ml of 0.1 M sodium phosphate, pH 7.2. Then 37.5 ml of hexadecyltrimethylammonium bromide (cetavlon; also named cetrimide) were added to precipitate the CA. Cetavlon was prepared by dissolving 2.5 g of cetrimide in 100 ml of 2% NaOH. This solution was heated briefly at 40 °C before use until a uniform solution appeared with no particulates. The precipitate was collected by centrifugation at 10 000 × g for 15 min at 0 °C to yield a clear supernatant. The pellet was dissolved in 100 ml of 1 m
NaCl. Then three volumes of ice-cold EtOH were added, and the mixture was placed on ice for 15 min to precipitate the CA. The precipitate was collected by centrifugation at 10 000 × g for 15 min at 0 °C to yield a clear supernatant. The pellet was dissolved in sterile water using the least amount of water required and dialyzed in sterile water O/N at 4 °C with three changes. The sample was lyophilized to dryness and the weight of CA was determined as stated above. The pellet was dissolved in sterile water using the least amount of water required, and the percentage solution was recorded and listed on the storage vial labels. The vials containing CA were stored at −25 °C.
We isolated a previously unidentified bacteriophage (phage), the NST1 phage, by its ability to form plaques on SC12078 bacteria. Briefly, SC12078 was inoculated into tubes with 5 ml of LB medium containing 0.4% glycerol and 10 µg/ml chloramphenicol and grown O/N. In total, 10-fold serial dilutions were prepared that contained five different NST1 phage particle numbers including 102, 101, 100, 10−1 and 10−2. Particle number was based on 1 µl of phage stock containing approximately 107 phage particles. 200 µl of the O/N growth were mixed with 1 µl of the phage stock to make the 107 concentration of phage. Dilutions containing 180 µl of O/N bacterial growth were mixed with 20 µl of the next higher concentration of phage. These dilutions were plated by quickly mixing each into 3 ml of LB+glycerol top agar, 0.7% agarose at 55 °C. The mixtures were quickly poured onto LB+10 µg/ml chloroamphenicol plates. The plates were incubated upside down at 37 °C for 5 h. After incubation, the plates containing phage plaques were stored at 4 °C. This process was used both to isolate the phage initially and was repeated once per month to maintain the NST1 phage. Growth of this phage was performed by inoculating phage plugs in the presence of SC12078 O/N cultures. Briefly, SC12078 was inoculated into LB medium containing 0.4% glycerol and 10 µg/ml chloramphenicol and allowed to grow at 37 °C in a shaker incubator O/N at 230 r.p.m. In total, 15 ml of the O/N culture were inoculated into each 1.5 l of LB medium containing 0.4% glycerol and 10 µg/ml chloramphenicol in three 4 l flasks. These cultures were allowed to grow at 37 °C in a shaker incubator at 230 r.p.m to an OD600 between 0.12–0.67, for approximately 2–4 h. Each 1.5 l SC12078 culture was inoculated with 30 NST1 phage plugs and grown at 37 °C with shaking at 230 r.p.m O/N. Growth was continued to an OD600 of 4.5–4.7. The cultures were centrifuged at 4200 r.p.m at 4 °C for 5 min. The supernatants containing the NST1 phage were collected for use in purifying the full-length CAE and were stored at −80 °C. The pellets containing bacterial cells and debris were discarded.
Purification of the CAE
Phenylmethanesulfonylfluoride (PMSF) was diluted to a final concentration of 0.1 mm and was added to 4 l of NST1 phage supernatant and placed at 4 °C. Using an Amicon filter apparatus and the YM30 membrane according to manufacturer’s instructions, the volume was reduced from 4 l to 4ml at 4 °C. The 4ml retentate was centrifuged at 40 000 × g for 60min at 4 °C. The supernatant was dialyzed with 10 mm Tris HCl, pH 7.5, 0.1 mm PMSF O/N at 4 °C with three changes. A Q Sepharose Fast Flow column (Pharmacia, GE Healthcare), 10 cm high and 1.5 cm diameter, was equilibrated with 10 mm Tris HCl, pH 7.5, and 0.1 mm PMSF until the pH of the solution eluting from the column was 7.5. The dialyzed supernatant was loaded onto the equilibrated Q Sepharose column and washed with two column volumes of 10mm Tris HCl, pH 7.5, 0.1 mm PMSF, approximately 30 ml. The column was eluted using a linear gradient from 10 mm Tris HCl, pH 7.5, 0.1 mm PMSF (150 ml) to 200 mm Tris-HCl, pH 6.5, 0.1 mm PMSF (150 ml) collecting 4 ml fractions (75 fractions in total) at a flow rate of 7 ml per h. The fractions were tested for CA-degrading activity using a viscometer postdigestion of CA (see protocol below), and the fractions containing the activity were pooled. The pooled fractions were concentrated on a disposable Amicon filter by centrifugation according to the manufacturer’s instructions. The protein concentration of the Amicon retentate was determined (Micro BCA; Pierce), and the sample was electrophoresed on a 4–12% gradient polyacrylamide gel and stained with Colloidal Blue (Novex, Invitrogen). A 120 cm column containing Toyopearl HW-50F (Catalog Number 07453; TosoHaas, Tosoh Bioscience LLC, King of Prussia, PA, USA) resin was equilibrated with phosphate-buffered saline pH 7.3–7.4, 0.1 mm PMSF. The retentate was fractionated on the equilibrated Toyopearl HW-50F column (TosoHaas, Tosoh Biosciences LLC), collecting 1 ml fractions. The fractions were tested for CAdegrading activity, and fractions containing the activity were pooled. The pooled fractions were concentrated on a disposable Amicon filter by centrifugation according to the manufacturer’s instructions. The protein concentration of the Amicon retentate was determined, and the sample was electrophoresed on a 4–12% gradient polyacrylamide gel and stained with Colloidal Blue. Approximately 206 µg of CAE at single-band purity was produced by this method.
A Wells-Brookfield Cone Plate viscometer (Catalog # LVDV-1+CP) with a CPE-40 cone was used to measure viscosity. This set-up provides the most sensitive measurement of changes in viscosity in the smallest volume, 500 µl. Viscometer accuracy was checked by measuring the viscosity of the standard, mineral oil. A solution of 1.2 mg/ml of CA was prepared in 0.05 m potassium phosphate buffer, pH 6.5. CA is highly viscous. All other samples were also prepared in this buffer. Dilutions of CAE (or other test samples) were added to the CA solution using identical volumes of this solution for each test or control sample. Approximately, 545 µg of CA were used in each sample, except for measurements of plasmid DNA alone in which no CA was used. CA alone or CAE, diluted in buffer alone was used as controls. Samples containing no protein were also added to CA for controls. The samples were incubated at 37 °C for 1 h. The samples were cooled to RT for 10 min. The viscometry readings for all samples were performed at RT for 30 s at 100 r.p.m. Viscosity values are measured in CentiPoise (cP). Calculations are based on the fact that sterile water has low viscosity and a cP of 1.00 at RT, and the dynamic viscosity of water at 20 °C is 1.002. Samples containing CAE and CA showed a decrease in viscosity, for example; whereas, control samples showed no change in viscosity.
Amino-acid analyses and database searching
The single protein band for the CAE, 84 354 MW, was prepared for Edman degradation and mass spectrometry analyses by the Baylor College of Medicine Protein Core Facility (Houston, TX, USA). Briefly, for Edman degradation, the CAE was electrophoresed on an SDS–PAGE gel poured at least 24 h before electrophoresis. The gel system included 0.1 mm thioglycolate as a scavenger in the upper-running buffer. The gel was electroblotted onto a polyvinylidene fluoride membrane using a Trisglycine buffer at RT for 2 h at 300 mA. The buffer consisted of 25 mm Tris, 192 mm glycine and 10% MeOH, pH 8.3. The gel was rinsed with sterile water for 5 min and stained with 0.05% Coomassie Blue in 1% acetic acid and 50% methanol. The gel was then destained in 50% methanol for approximately 15 min until the background turned pale blue. The gel was rinsed for 10 min in sterile water. The band was cut from the gel using a clean, fresh scalpel blade, placed into a 1.5 ml eppendorf tube, and submitted to the Core Facility. For mass spectrometry analyses, the CAE was electrophoresed on an SDS–PAGE gel using standard conditions. The gel was stained with 0.05% Coomassie Blue in 5% acetic acid and 10% methanol for 30 min to visualize the protein band. The gel was destained with 5% acetic acid and 10% methanol to clearly visualize the protein band. The gel was rinsed in sterile water for 15 min. The protein band was cut from the gel using a clean, fresh scalpel blade, placed into a 1.5 ml eppendorf tube, and submitted to the Core Facility. In a separate tube, an equal amount of gel containing no protein was submitted as a control.
The Edman degradation provided amino acid sequences for the following peptide fragment: ANSYNAYVANGSQTA
The mass spectrometry data provided amino acid sequences for the following eight-peptide fragments:
In each of these peptide fragment sequences the amino acid leucine (L) may actually be either leucine (L) or isoleucine (I), the amino acid aspartic acid (D) may actually be aspartic acid (D) or asparagine (N), the amino acid glutamine (Q) may actually be glutamine (Q) or lysine (K) and the amino acid phenylalanine (F) may actually be phenylalanine (F) or oxidized methionine.
Peptide fragments were searched against the Non-Redundant Protein Database, excluding the eucaryota organisms. Peptides were also searched using TBLASTN version 2.2.1044
against the database from the NCBI including the GenBank, EMBL, DDBJ and PDB sequences excluding the EST, STS, GSS, environmental samples and HTGS sequences. The search parameters used the BLOSUM62 matrix with gap existence penalty of 11 and gap extension penalty of 1. Peptides were also searched using BLASTP version. 2.2.1044
against the NCBI, Non-Redundant Protein Sequence Database that included the Genbank CDS translations, PDB, SwissProt, PIR and PRF, excluding environmental samples. There were 2 316 421 sequences with 787 539 419 total letters. The search parameters used the PAM30 matrix with Gap open penalty of 9 and extension penalty of 1. The peptide sequences were also searched against the phage sequences using a local installation of BLASTP. Three independent searches were performed by Kim Worley (Human Genome Sequencing Center, the Baylor College of Medicine), the Baylor College of Medicine Protein Core Facility and by Wei Zhang and Florante Quiocho (Baylor College of Medicine).
DNA sequencing of the CAE ORF
Genomic DNA was prepared from the NST1 phage. On the basis of the amino-acid sequences for peptide fragments determined by mass spectrometry and Edman degradation listed above, the following 12 degenerate oligonucleotides (oligos) were designed for sequencing the CAE ORF from the NST1 genomic DNA (sequences listed 5′–3′):
These degenerate oligos were designed by Kim Worley and prepared by Operon Technologies (Qiagen) and used for initial sequencing. Sequencing of both strands of the CAE ORF was performed by Lark Sequencing (Houston, TX, USA).
Chymotrypsin digestion and other proteases
Proteolytic digestion of the CAE with chymotrypsin was performed at 4 °C O/N in 20 mm Tris-HCl, pH 7.0, 100 mm NaCl and 5% glycerol. The following proteases were also tested including elastase, endoproteinase GluC, thermolysin, trypsin and proteinase K; however, these proteases did not cleave the CAE.
Cloning of CAE
PCR amplification of the NST1 phage genomic DNA using Deep Vent DNA Polymerase (New England BioLabs, Ipswich, MA, USA) for the CAE ORF extending from amino acids 107–790 with inclusion of an Nterminal methionine followed by glycine and then 6 histidines in the forward primer was performed. An Nco
I digestion site was also included in the forward primer and an Xho
I digestion site was included in the reverse primer. The forward are reverse primer sequences were the following (listed 5′–3′):
- Forward: TAGACCATGGGCCATCATCATCATCATCATCTCGCGCCACTTGACCGCGCC
- Reverse: TAGACTCGAGTTAGATGTAAGCCACCGGGTAGGTAGC
The amplification product was digested with Nco
I and Xho
I and ligated in proper orientation into the cloning site of the similarly digested pET-28a-c(+) vector (Novagen) using the Clonables Ligation/Transformation Kit (Novagen). The ligation mixture was transformed into Novablue competent cells (Novagen) and plated onto LB+kanamycin (80 µg/ml) plates. CAE-positive clones were identified by restriction enzyme digestion analyses.
Large-scale preparation of CAE
The CAE-recombinant clone was transformed in the E. coli host, BL21(DE3) (Invitrogen). A small-scale culture was first grown O/N in LB medium containing 50 mg/l of kanamycin at 37 °C with shaking at 225 r.p.m. In total, 2 l of hyper broth medium containing 50 mg/l of kanamycin was inoculated with 24 ml of the O/N culture and grown at 37 °C with shaking at 225 r.p.m until the OD600 reached 0.5, approximately for 2–4 h. Then the temperature was changed to 16 °C, and growth was continued for 30 min. Isopropyl β-D-1-thiogalactopyranoside was then added to make a final concentration of 0.015 mm, and the culture was grown for an additional 20 h. The growth medium was centrifuged at 4800 × g for 15 min, and the pellet was stored at −80 °C.
CAE was purified on a Ni-NTA column (Invitrogen) that selectively bound the 6 His-tag at the N-terminus using the following procedure. A 20 ml bed volume Ni-NTA column was equilibrated with 200 ml of buffer A consisting of 20 mm Tris-HCl, pH 8.0, 0.25M NaCl, 10% glycerol, 10 mm imidazole, 0.1 ml/l of β-mercaptoethanol and 1 mm PMSF. The cell pellet from the large culture was thawed and suspended in 150 ml of buffer A. The cell paste was disrupted using the Microfluidizer Processor, Model M-110Y, according to manufacture’s instructions (Microfluidics Corporation, Newton, MA, USA). The mixture was centrifuged at 40 000 × g to obtain a clear supernatant. The supernatant was loaded onto the equilibrated Ni-NTA column, washed with 600 ml of buffer A, and then washed with 80 ml of buffer B consisting of 20 mm Tris-HCl, pH 8.0, 0.25 m NaCl, 10% glycerol, 125 mm imidazole, 0.1 ml/l β-mercaptoethanol and 0.1 mm PMSF. The CAE was eluted from the column with 80 ml of buffer C consisting of 20 mm Tris-HCl, pH 8.0, 0.25MNaCl, 10% glycerol, 500 mm imidazole, 0.1 ml/l β-mercaptoethanol and 0.1 mm PMSF. The eluate was added to an Amicon Ultra-15 centrifugal filter unit with a 30 k cutoff (Millipore) and centrifuged at 2800 × g at 4 °C to concentrate the eluate. Buffer D, the storage buffer, consisting of 20 mm Tris-HCl, pH 8.0, 0.25 m NaCl and 10% glycerol was added to the eluate to provide a final volume of 13 ml. This concentration process using the Amicon Ultra-15 Centrifugal Filter listed above was repeated three times. The CAE protein was stored in buffer D at 4 °C at a 15 mg/ml concentration.
Plasmid DNA preparation and digestion with CAE
All plasmid DNAs made for Super Clean DNA processing were initially purified from 2.5 l LB cultures using Qiagen EndoFree Plasmid Giga Kits according to manufacture’s instructions. In addition, it was noted that bacterial growths reach an OD600 of 4.0 at 11–12 h post-inoculation of the starter culture (Qiagen Plasmid Purification Handbook 07/99, page 60). Furthermore, the maximal amount of plasmid DNA is produced from growth to an OD600 of 4.0. However, bacteria continue to grow to an OD600 of 5.0 if left in the incubator longer, with no further production of plasmid DNA. Therefore, we inoculated the ‘O/N’ growth late in the evening and started monitoring the OD600 in the morning, about 10 h post-inoculation. All bacterial growth was ended at an OD600 of 4.0 measured accurately using a turbidity cell holder in a DU 600 spectrophotometer (Beckman). The plasmid DNA was dissolved in 4 ml of 0.05 m potassium phosphate buffer, pH 6.5. CAE was added to the Qiagen EndoFree purified plasmid DNA in a weight ratio of 1:10 CAE:plasmid DNA. This mixture was incubated at 37 °C for 3 h. Then the temperature was increased to 50 °C and the mixture was incubated for 21 h.
Purification of CAE-digested plasmid DNA
The CAE-digested plasmid DNA was prepared for boronic acid column chromatography. The mixture was phenol-extracted twice and centrifuged at 13 000 r.p.m. for 10 min at RT. The aqueous phase was transferred to a sterile eppendorf tube and extracted with chloroform. The mixture was centrifuged at 13 000 r.p.m. for 10 min at RT, and the aqueous phase was transferred to a sterile eppendorf tube. The plasmid DNAwas precipitated with two volumes of 100% cold EtOH and 1/10 volume of 3 m sodium acetate, pH 5.2. The sample was placed at −20 °C for 1 h. The sample was centrifuged at 13 000 r.p.m. for 15 min at 4 °C. The plasmid DNA pellet was washed twice with 1 ml of 70% EtOH at RT and centrifuged at 13 000 r.p.m for 5 min at 4 °C. The plasmid DNA pellet was air-dried and resuspended in 1 ml of 0.2M ammonium acetate, pH 8.8, at approximately 3 µg/µl concentration.
Poly-prep chromatography columns (0.8 × 4 cm; BioRad, Life Science Research, Hercules, CA, USA) were equilibrated by pipetting 3.7 ml of 50% aqueous slurry of immobilized boronic acid (Pierce). Then 8 ml of 0.2 m ammonium acetate, pH 8.8, were added to each column, pipetted to disperse the resin and allowed to flow through until a small amount of buffer remained at the top. This step was repeated again. The plasmid DNA was loaded onto the equilibrated boronic acid columns without exceeding 3 ml volumes of DNA and not greater than 9 mg of DNA for each column. 5 ml fractions were collected in 1.5 ml eppendorf tubes using 0.2M ammonium acetate, pH 8.8, as the running buffer. The OD260 was measured for each fraction. The fractions containing significant amounts of plasmid DNA were pooled and loaded onto a second boronic acid column equilibrated with 0.2 m ammonium acetate, pH 8.8. In total, 5 ml fractions were collected in 1.5 ml eppendorf tubes using 0.2 m ammonium acetate, pH 8.8, as the running buffer. The OD260 was measured for each fraction. The fractions containing significant amounts of plasmid DNA were pooled. In total, 1/10 volume of 3 m sodium acetate and two volumes of −25 °C stored 100% EtOH were added to the chilled plasmid DNA and incubated on ice for 15 min. The mixture was placed at −25 °C for 1 h. The tubes containing plasmid DNA were centrifuged at 13 000 r.p.m for 30 min at 4 °C. The EtOH was aspirated from each tube. The DNA pellets were washed with 70% EtOH at RT and centrifuged at 13 000 r.p.m for 10 min at 4 °C. The EtOH was aspirated from each tube. This 70% EtOH wash-step was repeated again. The DNA pellets were air-dried. The boronic acid columns were also regenerated for reuse in purifying identical plasmids by first eluting the bound material, polysaccharides, with 0.1 m formic acid and then washing with five column volumes of 0.1 m formic acid. The column was then rinsed and stored in 0.02% sodium azide according to the manufacturer’s instructions.
The plasmid DNA was then prepared for Macrosep clean up. The plasmid DNA pellets were resuspended in 14 ml of 10 mm Tris-HCl, pH 8.0, containing 0.1% zwittergent 3–14 detergent (catalog No. 693017; Calbiochem, EMD4 Biosciences). The plasmid DNA tubes were incubated at 37 °C for 15 min. This mixture was placed into a Macrosep 100 Centrifugal Concentrator unit with 300 k cutoff and processed according to manufacture’s instructions. The unit containing the mixture was centrifuged at 4500 × g for 1 h at RT to concentrate the plasmid DNA. The retentate was brought to a 14 ml final volume by adding 10 mm Tris-HCl, pH 8.0, containing 0.1% zwittergent as stated above. The mixture was centrifuged at 4500 × g for 1 h at RT. This process was repeated three more times without inclusion of the zwittergent in the final two concentration steps. The plasmid DNA was precipitated with two volumes of 100% cold EtOH and 1/10 volume of 3 M sodium acetate, pH 5.2. The tubes containing plasmid DNA were placed at −20 °C for 1 h and then centrifuged at 13 000 r.p.m for 15 min at 4 °C. The DNA pellets were washed twice with 1 ml of 70% EtOH at RT and centrifuged at 13 000 r.p.m for 5 min at 4 °C. The EtOH was aspirated from each tube. The DNA pellets were airdried, resuspended in sterile water at a final concentration of 5 mg/ml and stored in nunc vials at −85 °C.
This BCA assay was performed to detect any residual reducing ends from polysaccharides remaining in the Super Clean processed plasmid DNA. However, it can also be used to determine the activity of CAE. A modified version of the published, miniaturized highly sensitive BCA assay was used.26,27
Briefly, this assay used an optical 96-well reaction plate (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), with each well containing a mixture of 2 µg of CAE (test sample) or bovine serum albumin (control sample) and 100 µg CA or plasmid DNA in a total volume of 110 µl of 0.05 m
potassium phosphate buffer, pH 6.5. All wells were sealed with strip caps, and the plates were centrifuged at 1500 r.p.m at RT for 1 min. The caps were removed from the wells, and the solution mixed in each well by gently pipetting up and down three times using a multi-channel pipettor. The wells were resealed using strip caps and incubated at 37 °C for 3 h and then at 50 °C for 21 h. The plates were centrifuged at 1500 r.p.m at RT for 1 min. Then 100 µl of each reaction mix was transferred to a new 96-well plate using a multi-channel pipettor and mixing gently by pipetting three times up and down. Then 100 µl of freshly prepared BCA reagent (MicroBCA Protein Assay Kit; Pierce) were added to each well. Lids were placed over the plates and incubated at 37 °C for 2 h, cooled to RT for 15 min, and read on a multi-plate reader at 550 nm. Each test or control sample was assayed in triplicate. The results were determined by subtraction of any values obtained for the control sample from the test sample.
Liposome and complex preparation
The BIV liposomes and complexes were prepared as previously described except that synthetic cholesterol (Sigma) was used in place of extracted cholesterol at a DOTAP:cholesterol ratio of 50:45. The complexes were filtered through a 1.0 µm pore size, polysulfone filter of 13 mm diameter (catalog No. 6780-1310; Whatman, GE Healthcare) before the administration into mice.
In vivo toxicity assay
Mice were IV injected into the tail vein slowly and evenly over one minute using a 30-gauge syringe needle. Male Balb/c mice were purchased from Harlan Laboratories (Houston, TX, USA). The SCID mouse strain used was HsdIcr:Ha(ICR)-Prkdcscid bred in the Barrier Animal Facility, Baylor College of Medicine. Human pancreatic PANC-1 tumor-bearing mice were established in these SCID mice. The PANC-1 cells were purchased from the (Manassas, VA, USA) ATCC. The PANC-1 cells were harvested and resuspended in 1 × phosphate-buffered saline. A 200 µl cell suspension containing 5 × 105 PANC-1 cells was intraperitoneally injected into each 8–10 week-old male SCID mouse. Tumors were allowed to grow for 7 weeks before IV injections. All animal procedures were performed in accordance with the Baylor College of Medicine institutional guidelines using an approved animal protocol.