Gel Filtration on Sephacryl

Apply the concentrated component I to a column of Sephacryl S-300 (2.5x95cm) equilibrated with 50 mM PB, pH 7.5, and elute with the buffer. Collect the fractions containing component I and concentrate by ultrafiltration as described above. Check the purity of the component using SDS-PAGE. If the preparation contains any impurities, run the gel filtration again on the column of Sephacryl S-300 under the same conditions as above. 

By these purification procedures, 1-3 mg of component I and 20-40 mg of component II are recovered from 4 I of culture of C. botulinum type C strain 92-13. The mixture of untrypsinized component I and trypsinized component II has high lethal activity in mice (specific activity is about 2x10 mouse intraperitoneal 50% lethal doses per mg of protein), although each of these components alone shows very low activity even after trypsinization. The purified components I and II each show one band in SDS-PAGE, and their molecular weights as determined by the electrophoresis are 45kDa and 100 kDa, respectively. 

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The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Gel filtration on Sephacryl S-200 was employed. Polymers whose molecular weights had been measured by light scattering photometry were run on a Sephacryl column and the elution volumes... 

Gel-filtration and affinity chromatography For separation of the ALA-synthesizing components we used gel-filtration on Sephacryl S-300 and subsequent affinity chromatography as a sequential system of Blue-Sepharose, Red Agarose and Chlorophyllin Sepharose (7). 

On Sephacryl S-300 (Fig 1) GSA-aminotransferase and tRNAGlu-ligase (fraction 30-55) were separated from membrane fragments (fraction 1-20), low molecular weight components (fraction 60-100). The elution profile and activity-pattern of gel-filtration on Sephacryl S-300 was very similar to that of the unicellular green alga Scenedesmus obliquus (15) and barley (16). 

Figure 4 Coelution of the labeled soybeana-CT2, imported into isolated pea chloroplasts, with the pea ACCase activity during gel-filtration on Sephacryl S 300.

To test the functionality of the in vitro synthesized subunits we performed the fractionation of clarified pea stroma containing imported soy proteins by gel-filtration on Sephacryl S300. We found that the labelled soy proteins coelute with ACCase activity in high MW range (Fig.4), which we think is the evidence for their integration into pea ACCase. This supports our assumption that the discussed soy accA and accB genes are coding for the components of soybean chloroplastic ACCase complex,... 

Figure 8.22 The activities of exopolyphosphatases 1 ( ) and 2 ( ) in the cytosol fraction of Saccharomyces cerevisiae during the process of growth ( ) at a low initial cell density without reinoculation (a) complete Reader medium (b) P -limited medium. The cytosol fraction was subjected to gel filtration on a Sephacryl S-300 column. The exopolyphosphatase activities were estimated separately in the fractions corresponding to the molecular masses of 40 kDa (exopolyphosphatase 1) and 830 kDa (exopolyphosphatase 2).

The bound MAb is eluted with 25 mM acetic acid and after neutralization is subjected to gel filtration on a column of Sephacryl S-200 HR (length 60-90 cm) equilibrated with PBE containing 0.15 M NaCl (PBS) at pH 7.5. 

Antibodies modified with SPDP (2.5 PDP groups/IgG) are reacted with 2-IT-treated PAP (see Subheading 3.2.) after excess SPDP and 2-IT are removed by gel filtration on Sephadex G-25M. The PAP /IgG molar ratio is 3 1. After incubation at 25°C for 2 h the mixture is chromatographed on a TSK-3000-SW column (HPLC) or a Sephacryl S-200 HR (gel filtration) column, both equilibrated with 100 mM phosphate buffer, pH 6.8. The fractions containing IT and the unreacted IgG are further chromatographed in columns of CM-Sepharose equilibrated in 10 mM phosphate buffer, pH 6.2. At this pH all the free IgG is washed out, whereas the bound IT is eluted by increasing the pH to 7.8 and adding 20 mM NaCl to the phosphate buffer. The purification scheme is presented in Fig. 9. 

In order to remove the residual PME the AE fraction was further fractionated on Mono S cation exchange column. Unlike the DEAE-Sepharose column, where PME elutes after the AE activity, the order of elution was reversed on the Mono S column (Fig. 1). The last step in the purification was a gel filtration (Sephacryl S-200) column. 

Unlike bovine serum amine oxidase, which is available in gram quantities and was used as the prototypic system for the establishment of TPQ, a typical preparation of LO yields about 5 mg (starting with about 500 grams of aorta tissue). In an effort to minimize protein loss, purification was stopped after the stage where LO (32 kDa) coelutes with a second protein (24 kDa) from Sephacryl S-200 gel filtration. The two-banded protein was labeled with [ 4C]phenyl-hydrazine, to yield the expected chromophore for a phenylhydrazone derivative of a quinone structure (Fig. 1). Analysis of the i C-labeled protein by SDS gel electrophoresis showed almost exclusive incoiporation of into the 32-kDa LO band (13). Therefore, the unreactive 24-kDa band would not affect subsequent procedures since selection of active site-derived peptides from LO was dependent on screening for in addition to monitoring die UV-Vis absorption of the newly formed chromophore. 

So far, it has not been possible to preserve the activated component II by freeze-drying. Therefore, it is desirable to prepare the activated component II at the time of use and to store it in the low temperature freezer (-80 °C) in vials. A gel-filtration pattern of trypsinized component II on Sephacryl S-300 and SDS-PAGE of the fractions are shown in Figure 1 in this case, one protein peak of activated component II was obtained. 

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