Protamine fractionation

Zeins, the major storage proteins of maize belonging to the prolamins. In maize, the ethanol-soluble or protamine fraction consists of the zeins (Mr 10-22 kDa) that constitutes as much as half of the total protein of the endosperm. They are characterized by a high content of Glu (23%) and Leu (19%). The amino acid sequence of a representative of the zeins has been derived from the nucleotide sequence of a zein cDNA clone [D. Geraghty et al.. Nucleic Acids Res. 1981, 9, 5163]. 

To avoid the use of mineral acids, Kossel and Schmiedeberg (see Kossel, 1929, p. 19) devised the use of cupric chloride (or sulfate) to extract protamine from sperm heads. Dry sperm heads are treated with aqueous cupric chloride (or sulfate) for several days at room temperature. During the process, due to double decomposition, the protamine moiety is transferred into the aqueous phase in the form of hydrochloride (or sulfate) salt, while the nucleic acid moiety is converted to the insoluble cupric salt after removal of the insoluble salt, the protamine fraction is precipitated from the solution as picrate salt. 

It has been reported that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing NLS. NLS are short karyophilic peptides on proteins that bind to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus. Examples of these peptides will be given later in this section. DNA can also be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions (110), cationized albumin, and others. These molecules increase the transfection efficiency. In addition histone HI is identified as transfection-enhancing protein in cell culture (111). 

A large scale preparation of E. coli 045 was subjected to enzyme purification using the assay for 3,5-epimerase. Protamin sulfate precipitation, ammonium sulfate fractionation was followed by DEAE-chroma-tography. The fraction containing enzymatic activity, as measured by tritium exchange, was eluted from the DEAE column early. This fraction was incapable of producing any net synthesis of TDP-6-deoxy-L-... 

This chapter will focus on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in Fig. 1. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins (Table 2). Reduction in volume, removal of major nonprotein... 

FIGURE 14 Purification of rHu-BDNF from its variants by displacement chromatography. (Barnthouse et a/.39) (A) Analytical chromatography of feed stock by high temperature reversed-phase acetonitrile gradient. Column 4.6 X 250 mm Vydac C4 sample 50 jj.% injection of rHu-BNDF. (B) Displacement of rHu - BDNF by protamine. Column 4.6 X 500 mm, POROS HS / M loading 20 mg / mL column volume mobile phase 50 mM phosphate buffer, pH 7.0, 500 m/W NaCI displacer 2 mM protamine sulfate. (C) High-temperature reversed-phase assay of three displacement fractions. Overlay of fractions from the early (fraction 16), intermediate (fraction 18), and late (fraction 31) parts of the displacement train. 

Adverse effects. When bleeding is induced by heparin, the heparin action can be instantly reversed by protamine. Against fractionated heparins and fondaparinux, protamine is less or not effective. Heparin-induced thrombocytopenia type II (HIT II) is a dangerous complication. It results from formation of antibodies that precipitate with bound heparin on platelets. The platelets aggregate and give rise to vascular occlusions. Because of the thrombocytopenia, hemorrhages may occur. Fondaparinux is also contraindicated in HIT II. 

Acetyl-CoA arylamine N-acetyltransferase was purified from pigeon liver by using steps involving protamine sulfate, ammonium sulfate fractionation, and affinity chromatography on immobilized amethopterin. Figure 9.20 shows a representative chromatogram. In another study, Thomas et al. (1990) used tryptamine as the substrate. In addition, whereas the study by Manneus et al. (1990) appeared to require pure enzyme, Thomas s study did not. 

Figure 12.24 Displacement chromatogram of a protein mixture. (A) Experimental results. 10 X100 mm Protein-Pak SP-8HR cation exchange column eluent, 12.5 mM Na2P04 displacer, 15 mg/mL purified protamine. Feed, 10.78 mg a-chymotrypsinogen A, 7.86 mg cytochrome c, and 17.3 mg lysozyme in 1.7 mL. Fp 1 mL/min, 100 pL fractions. (B) Displacement chromatogram calculated with the ideal and the steric mass action models. Reproduced with permission from J. Gerstner and S. Cramer, Biotechnol. Progr., 8 (1992) 540 (Fig. 4). 1992, American Chemical Society.

H2O, protamine precipitation, (NH4)2S04 fractionation, Me2CO precipitation, acid treatment at pH 5.7 and precipitation at pH 4.5. The equilibrium constant for pyruvate + -acefyl-D-mannosamine A-... 

Purification ammonium sulfate fractionation, Q-Sepharose, heat treatment, HIC (phenylsepharose, preparative gel electrophoresis) heat treatment, Sephadex G-50, DEAE-cellulose, HIC (phenylsepharose), Fractogel DEAE-Sephacel, HIC (phenylsepharose), SEC (Sephacryl S-200 HR), Mono Q and superose-12 protamine sulfate and ammonium sulfate precipitation, heat treatment, IEX (DEAE-Sephadex and Trisa-cyl, HIC (octyl-Sepharose)... 

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