◆ SpookStack

United States Patent: 6,387,665 e Page 8 of 11 * chosen and scanned to determine background absorption for an accurate baseline. The region to be scamned 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 ona 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 KCI 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 KCl. 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.mu. 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 a linear gradient to 30% of the 1 M NaCl DEA elution buffer. The purified PA was found to elute between 10-15%, while the smaller molecular weight proteolytic breakdown products eluted as a shoulder or partially resolved peak at 16-20% of the elution buffer. The resolution of the two peaks was found to be a function of content of PA proteolytic degradation products. The eluant was monitored at 280 nm and peak fractions were collected by manual triggering of an ISCO fraction collector. Samples of the peak fractions were diluted into 5-10 volumes of TRIS pH8.0, 0.1 mM PMSF, 50 AM OP, 1 mM EDTA buffer and concentrated using Amicon Centricon 30 concentrators at 4500.times.g at 4.degree. C. to approximately the initial sample volume. An equal volume of SDS-PAGE solubilization buffer was added to the sample immediately prior to heating at 95.degree. C. for 5 min. Purity was assessed from 8-25% SDS-PAGE PHAST gels (Pharmacia) and fractions with the highest purity were combined and dialyzed against 40- 50 volumes of 25 mM DEA pH8.9, 50 mM NaCl, 0.1 mM PMSF and 2 mM EDTA at 4.degree. C. for at least 16 hrs. Fractions judged empirically to be less than 95% pure were rechromatographed under the http://patft.uspto.gov/netacgi/nph-Parser?Sectl1=PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005