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United States Patent: 6,316,006 Page 7 of 10 . © @ * diluted 1:1 with a 2.times.SDS solubilization buffer consisting of 50 mM Na.sub.2 CO.sub.3, 4% (w/v) SDS, 12% (v/v) glycerol, 2% (v/v) 2-mer-captoethanol and 0.01% (w/v) Bromphenol Blue prior to heating at 95.degree. C. for 5 min. The fermentation samples containing varying amounts of PA 83 kDa were solubilized as described above and run on a Daiichi 4-20% gradient TRIS/TRICINE gel to approximate total yield of PA. Two hundred to 2000 ng samples of purified PA were solubilized in the same buffer and loaded onto the gel in constant total volume of 3 .mu.l. Three or four appropriate dilutions of the fermentation samples determined from the first gel were loaded onto the gel with the standards and electrophoresed at 100 V initially and 140 V once the samples entered the separating gel and until the Bromphenol Blue dye reached the bottom edge of the separating gel. The gel was then fixed in 10% (v/v) acetic acid 20% (v/v) MeOH for 10 min, rinsed with MQ H.sub.2 O and stained with Coomassie Brilliant Blue 0.05% (w/v) in 10% (v/v) acetic acid for a minimum of 16 hrs to allow complete and uniform staining. The stained gel was then destained in 10% (v/v) acetic acid until the background contained no visible residual dye. The gel was then scanned on a laser densitometer (LKB, Ultrascan XL Laser Densitometer). Representative portions of the gel without protein were randomly chosen and scanned to determine background absorption for an accurate baseline. The region to be scanned for each lane containing PA was then visually aligned to insure that the entire protein peak and adequate baseline were included in each scan. The scans were completed and the integration values were determined using the LKB preprogrammed Gaussian algorithm and later were confirmed by cutting out individual peaks and manually integrating based on peak weight. The resulting integration values were plotted using Sigmaplot (Jandel). Linear regression of the results revealed typical r values of 0.992- 0.996. The linear standard curve was then used to quantitate the amount of 83 kDa PA in the various fermentation samples based on the same integration methods. Purification: The exact volume and conductivity of the PA in DEA buffer was determined and solid KCI was added to the solution to a final concentration of 30 mM and conductivity of 10-11 mmhos/cm. The PA was pumped with a peristaltic pump through a monoQ column prepared by collecting 100 mls of hydrated Bio-Rad Macro Prep 50Q on a sintered glass filter and washing sequentially with 1 liter of 25 mM DEA, 50 mM NaCl, 1 mM EDTA, 50 .mu.M OP and 0.1 mM PMSF pH8.9 and 1 liter of the same buffer with 30 mM KCl added. The conductivity (10-11 mmhos/cm) and pH of 8.9 of the eluate from the Macro Prep 50Q after the second wash were comparable to that of the PA solution after addition of KCI. The Macro Prep 50Q resin was then degassed and slurry packed into a Pharmacia K column with a Rainin Rabbit-Plus peristaltic pump at 48 rpm and a flow rate of 15 mls/min. The final column volume was (5.times.5 cm) 98 mls. The PA solution was pumped through the Macro Prep 50Q column at a rate of 10 mls/min and the eluate was collected until all of the PA sample volume was loaded and the column washed with an additional 100 mls of DEA/KCI buffer. The eluate containing unbound PA was concentrated and diafiltered using an 1-ft.sup.2 30 kDa cutoff cellulosic-Amicon wound spiral cartridge at an operating pressure of 20 psi. The final concentrate (ca. 400 mls, 6-7 mmhos/cm) was passed through a 0.2 A cellulose acetate filter. The filtered PA was loaded onto a Poros IIQ perfusion chromatography column using a quaternary Waters 600E HPLC pump. The column was prepared by hydrating seven grams of the Poros IIQ perfusion resin in twice the packed bed volume of 2% (w/v) NaCl. After settling the resin was resuspended in six times the packed bed volume of 25 mM DEA pH 8.9, 50 mM NaCl, 7.5%(v/v) ethylene glycol and allowed to settle overnight at room temperature. The resin. was then resuspended in three times the packed bed volume and finally in one and one-half times the final volume before the slurry was extensively degassed using a vacuum pump (vacuum unknown). The entire degassed slurry was then transferred to a Waters AP 20.times.100 mm glass HPLC column and the column was packed in one step using the Waters 600E pumps at a flow rate of 20 mls/min and a backpressure of 650 psi at room temperature. The column separation efficiency was then tested at a flow rate of 10 mls/min using a linear 1 M NaCl gradient and ovalbumin 5 mg/ml (Sigma) and bovine serum albumin 10 mg/ml (Sigma) in DEA as buffer as standard proteins. Approximately 100 mls of PA (ca. 20-30 mg PA) cooled to 4-6.degree. C. was applied to the column and followed with a 20 min wash in the starting buffer at room temperature to elute unbound material. The column was then developed with hitp://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005