CM-Sepharose

The dialysed sample was fractionated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose CL-6B column (1.5 x 15 cm). Unbound proteins were removed with 50 mM MES pH 6.8, 1 mM DTT, and the bound proteins were eluted with an increasing NaCl gradient from 0 - 0.4 M NaCl in a total volume of 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein profile was measured at 280 nm. 

AE was purified from orange peels. After homogenization, precipitation with 30 - 60% (NH4)2S04 followed by dialysis, the sample was applied to a cation exchange column (CM-Sepharose CL-6B). AE binds strongly to a cation exchange column material at pH 6.8... 

Curculin which is extracted with 0.5 M sodium chloride from the fruits of Curculigo latifolia and purified by ammonium sulphate fractionation, CM-sepharose ion-exchange chromatography and gel filtration.The protein acts as a low calorie sweetener and has a maximum sweetness equal to 0.35 M of sucrose. In addition to its sweetness, curculin also has taste modifying abilities since water and sour substances elicit a sweet taste after consumption of curculin. Currently, there is no other protein that has both sweet taste and taste modifying abilities. 

Enzyme Purification. The purification of the xylanolytic enzymes began with adsorption on a cation exchanger (CM-Sepharose FF) at pH 4.0. The final purification was accomplished by another ion exchange step as described previously for xylanase (24), / -xylosidase (25), a-arabinosidase (26) and acetyl esterase (27). 

Figure 11. Effect of incubation time (at 37°C) and of ganglioside concentration on the incorporation of gangliosides (G.y, G m, GTib) into phosphatidylcholine mono-lamellar vesicles. Phosphatidylcholine (as vesicles) 9 nmol. Ganglioside from 0.5 to 2 nmol. After incubation the mixtures were passed through a 1 X 20 cm Sepharose 4B column to separate vesicles from ganglioside micelles.

Crude enzyme extract (20 mM potassium phosphate buffer, pH 5.5, 1.8 mL, 17.9 units) was applied to a column of CM-Sepharose CL 6B which was previously equilibrated with 20 mM potassium phosphate buffer, pH 5.5. Non-adsorbed protein was washed off with 20 mL... 

Table 1. Purification of EcaL-ASNasc using a two-step procedure employing cation-exchange chromatography on CM-Sepharose and affinity chromatography on immobilized L-Asn. The procedure was carried out at 4°C...

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

The combined culture supernatant and cell wash are centrifuged at 16,000 g, 4°, for 20 min to remove residual cells and debris. The supernatant is loaded onto a 1.9 X 14 cm column containing 30 ml of CM Sepharose Fast Flow resin (Pharmacia, Piscataway, NJ) previously equilibrated in KP 6.2 at 4° using a peristaltic pump at a rate of approximately 10 ml/min. 

The supernatant was subjected to fractional precipitation with ammonium sulfate (step 5) and then with acetone (step 6). PTTH was recovered in the precipitates with 35-55% acetone, while bombyxins were recovered with 55-75% acetone. The 35-55% acetone precipitates were subsequently purified through five steps of conventional chromatography gel filtration on Sephadex G-50 with 0.5M Tris-HCl (pH 8.5) (step 7), anion exchange on DEAE-Sepharose C1-6B with 0.2M sodium acetate (pH 5.2)(step 8), cation exchange on CM-Sepharose C1-6B 0.1-0.5M NaCl in 0.05M sodium acetate (pH 5.2) (step 9), Hydrophobic adsorption on Octyl-Sepharose C1-4B with 4M ammonium acetate, 0.2M ammonium acetate and 40% acetonitrile in 0.2M ammonium acetate (step 10) and gel filtration on Sephadex G-75 with O.OIM phosphate buffer containing 0.2M NaCl and 2% butanol (step 11). 

The supernatant is diluted with an equal volume of 20 mM HEPES NaOH, pH 7.5 (buffer A), and applied to a CM-Sepharose column (2.5 cm i.d., x 10 cm) equilibrated with buffer A. The column is washed with 100 ml of buffer A. Elution is performed by a linear gradient between 0 and 0.5 M NaCI in buffer A. 

From Pharmacia (Piscataway, NJ) Protein A-Sepharose Fast Flow, Protein G-Sepharose Fast Flow, Sephacryl S-200HR, DEAE-Sepharose CL-4B, Sephadex G-25M, Blue-Sepharose CL-4B, Sephadex G-25 MicroSpin, CM-Sepharose CL-4B, SP-Sepharose Fast Flow. 

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. 

Another new technology that offers promise for commercial biotechnology purifications is the use of parametric pumping with cyclic variations of pH and electric field. This has been described by Hollein and cowork-ers.fi26) They worked with human hemoglobin and human serum albumin protein mixtures on a CM-Sepharose cation exchanger. The extensive equations they reported for parametric separations allow analysis of other systems of two or more proteins which may be candidates for this type of separation. 

Use a cation-exchange column, such as CM-Sepharose, and run it at pH 6. Protein X will have a positive charge and will stick to the column. 

Fig. 2. Gel exclusion chromatography of chro-matophore preparations on a Sepharose 2B column. Chromatography was performed essentially as described by Fraker and Kaplan (18). A 0.1 ml sample was loaded onto a 1.7 x 35 cm Sepharose 2B column the flow rate was 11 ml hr Sodium phosphate buffer (100 mM), pH 7.5, containing lOmM EDTA and 0.02% soium azide was used for both equilibration and elution. The effluent was passed through an LKB 2138 Uvicord S detector to...

Purified lAP was an oligomeric protein with a Mj. value of 117,000. It was ahexamer composed of five dissimilar subunits, Si (M = 28,000), S2 (23,000), S3 (22,000), S4 (11,700), and Ss (9,300). Exposure of lAP to 5 Af urea at 4°C for 3-4 days gave four separate protein peaks upon the subsequent column chromatography with CM-Sepharose the two were Si and Ss and the other two peaks were dimers (Di and D2). These two dimers were further separated by exposure to 8 Af urea for 16 h followed by DEAE-Sepharose chromatography to the constituent subimits Di to S2 and S4, and D2 to S3 and S4. Thus, these five subunits were separated from each other and purified to homogeneity as revealed by a sharp single peak on polyacrylamide gel electrophoresis [21]. Based on the relative color intensity of the individual subunit stained on sodium dodecy sulfate (SDS)-polyacrylamide gel, the molar ratio of these subunits was calculated as 1 1 1 2 1 in the native lAP molecule. 

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