Solubility protein crystallization

The bacterial culture converts a portion of the supplied nutrient into vegetative cells, spores, crystalline protein toxin, soluble toxins, exoenzymes, and metabolic excretion products by the time of complete sporulation of the population. Although synchronous growth is not necessary, nearly simultaneous sporulation of the entire population is desired in order to obtain a uniform product. Depending on the manner of recovery of active material for the product, it will contain the insolubles including bacterial spores, crystals, cellular debris, and residual medium ingredients plus any soluble materials which may be carried with the fluid constituents. Diluents, vehicles, stickers, and chemical protectants, as the individual formulation procedure may dictate, are then added to the harvested fermentation products. The materials are used experimentally and commercially as dusts, wettable powders, and sprayable liquid formulations. Thus, a... 

Its solubility characteristics in aqueous systems are such that retention of toxicity to insects by dissolved crystal protein is always suspect, and loss of activity on dissolution owing to denaturation is often observed. The protein is soluble only in relatively strong aqueous alkali. Thus, it has been variously reported to be soluble in 0.01N- to 0.05N sodium hydroxide (1) and alkali at pH 10.5 in the presence of thioglycollate (35) we have also observed its solubility in alkali at pH 9.5 in the presence of urea and potassium boro hydride. One difference between the characteristic proteins produced by various strains of crystalliferous bacilli is observed in the degree of alka-... 

Polymorphism and solvatomorphism are not, of course, limited to small molecules, and such phenomena can be observed in protein crystals as well. Two polymorphic forms of aprotinin have been identified, and the solubility of these studied in a variety of aqueous media [84], The needle polymorph was found to exhibit increased solubility with increased temperature (i.e., an endothermic heat of solution), while the solubility of the bipyramid form decreased by with increasing temperature (i.e., an exothermic heat of solution). The solubility curves crossed at 25 °C for a pH of 4.75, and hence one could obtain the desired crystal form through a judicious selection of crystallization temperature. 

Proteins crystallized from very low salt concentrations (examples are carboxypeptidase A and elastase) can often be treated exacdy like proteins crystallized from alcohol-water mixtures. Their low solubility in water allows them to be transferred from their normal mother liquor to a distilled water solution or to a solution of low (10-20%) alcohol concentration without disorder. It is advisable to carry out this transfer at near 0 C to further decrease the protein solubility. From this stage it is trivial to add alcohol while cooling, as described above. Complications arise, however, when the salt employed as a precipitant in the native mother liquor is insoluble in alcohols. The solution to this problem is to replace the salt by ammonium acetate at equivalent or higher ionic strength. Ammonium acetate is soluble up to 1 M in pure methanol, and is very soluble in nearly all alcohol-water mixtures, even at low temperature. It therefore provides a convenient substitute for salts such as sodium sulfate or sodium phosphate. 

The fastest way to obtain co-structures with a protein and fragments is to soak the fragments into existing crystals. Since each protein is unique, trial and error will be necessary to deduce the conditions where your protein crystals are stable and the fragments are suitably soluble (referred to as the protein stabilization buffer) (rrrNote 9). 

Carsten Jacobsen (Novo Nordisk) presented results on protein crystallization in preclarified, concentrated fermentation broths. In particular, the impact of filtration rate on the formation of favorable large diamond versus rod shapes was examined. By adding seed crystals just above the solubility curve, where no nucleation occurred, the authors were able to produce 30% larger crystals as compared to an unseeded crystallization. Although there was minimal recovery and characterization data, this technique may prove very beneficial for dealing with difficult feed streams. While the work presented in this talk was done at the laboratory scale, scale-up experiments will be required to confirm the suitability of this approach for industrial process applications. 

The existence of -barrels was established for chymotrypsin at a very early stage in the now common protein crystal structure analyses. This enzyme contains two distorted six-stranded -barrels with identical topologies (Birktoft and Blow, 1972). A selection of -barrels in water-soluble proteins is given in Table I. The very abundant TIM-barrel consisting of eight parallel /1-strands was also detected rather early (Banner et al., 1975). Additional eight-stranded /1-barrels of this group are those of streptavidin (Hendrickson et al., 1989) and of the lipocalins (Newcomer et al., 1984). 

Crystal solubilization is facilitated by an alkaline pH of susceptible insects. The typical midgut pH is between pH 9-11 in lepidopteran larvae [37-39]. In mosquito larvae, the pH inside the posterior midgut/gastric caeca is between 7-8, while the pH inside the anterior midgut is close to 11 [40]. Thus alkaline buffers are usually used for in vitro solubilization of lepidopteran and dipteran active B. thuringiensis crystals. Differential crystal solubility can be useful in partial separation of toxins. For example, CrylA toxins are fully soluble at pH 9.5, while the Cry2 proteins require a pH of 12 for complete solubilization [41]. Moreover, pH has different effects on Cry toxin pore-formation activities [42], and differences in the level of solubilization can contribute to toxicity differences... 

The growth of protein crystals is a difficnlt, complex, and often frustrating procedure. The protein crystal is precipitated from a snpersatnrated solntion of the macromolecule in which the protein is partitioned between the solid phase and the solntion. The pH value influences the solubility. Usually a pH is chosen near the isoelectric point of the macromolecule. Inorganic salts, organic solvents, and commercially available precipitating agents, such as the polymer PEG, can be helpful. 

Membrane protein crystals have significantly more solvent (64%) content than soluble proteins [47% (44)] presumably because of the detergent in the crystal. The orgaiuzation of the detergent in the membrane protein crystal has been investigated in a select few cases and is different in each case. In the LH2 crystal, the detergent forms a belt around the hydrophobic surface of the protein consistent with the dimension of the OG... 

Precipitant A chemical used to promote protein crystallization, but not denat-uration. Examples are highly soluble inorganic salts (ammonium sulfate or sodium chloride), and organic polyethers (polyethyleneglycols of a selected molecular weight range). 

D.W. Bolen, Effects of naturally occurring osmolytes on protein stability and solubility issues important in protein crystallization, Methods 34 (2004) 312-322. 

B. Guo, S. Kao, H. McDonald, A. Asanov, L.L. Combs, W.W. Wilson, Correlation of second viral coefficients and solubilities useful in protein crystal growth, J. Cryst. Growth 196 (1999) 424-433. 

In the last 10—15 years, a surge in the interest on both experimental and theoretical features of the OSVC of a protein (2) in mixed water (1)—cosolvent (3) mixtures has taken place. This surge was caused by the connection between OSVC and protein crystallization as well as its solubility in water and in aqueous mixed solvents. 

Second, it was suggested that the solubility of a protein in aqueous mixed solvents can be correlated with the osmotic second virial coefficient. However, whereas the connection of B22 to protein crystallization was widely accepted, the connection of B22 to the solubility of proteins requires additional investigation. 

As with pH, proteins may vary in solubility as a function of temperature, and some are quite sensitive. One can take advantage of this property with both bulk and microtechniques (Jacoby, 1968 McPherson, 1999). Many of the earliest examples of protein crystallization were based on the formation of concentrated solutions at elevated temperatures followed by slow cooling. Osborne in 1892 successfully crystallized over 20 plant seed globulins by cooling relatively crude extracts from 60°C to room temperature in the presence of varying concentrations of sodium chloride. 

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