Purified Proteins

Many laboratory techniques have been described to purify proteins (25), but they are often too cosdy for industrial enzymes, especially column separations. However, aqueous two-phase extraction (26) and ion exchange are used. 

Before protein molecules attain their native folded state they may expose hydrophobic patches to the solvent. Isolated purified proteins will aggregate during folding even at relatively low protein concentrations. Inside cells, where there are high concentrations of many different proteins, aggregation could therefore occur during the folding process. This is prevented by... 

TABLE 3.8 Recovery of Purified Proteins on Zorbax GF-250 Columns... 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

M NaCl was 15 hr at 0°C and 45 min at 23°C. Based on this inactivation rate, it is estimated that the purified protein contained only 5-10% of the active form. No method has been found to stabilize the photoprotein. 

Neuronal NO synthase (nNOS) is constitutively expressed in neurons of the brain. Its activity is regulated by Ca2+ and calmodulin. Half-saturating L-arginine concentrations are around 2 pM. cDNAs encoding nNOS have been cloned from rat and human brain. The open reading frame of human nNOS consists of 4299 bp, corresponding to 1433 aa. This predicts a protein of 160 kDa, which is in accordance with the molecular mass of the purified protein. 

Persons with acquired immunodefidency syndrome or those who are positive for the human immunodefidency virus and have a positive tuberculosis skin test or a negative tuberculosis skin test but a history of a prior significant reaction to purified protein derivative (a skin test for tuberculosis)... 

A sample consisting of 155 mg of a purified protein is dissolved in 10.0 mL of ethanol. This solution is placed in a device for measuring osmotic pressure and rises to a final height of 32.5 cm above the level of pure ethanol. The experiment is performed at 1.00 atm and 298 K. The density of ethanol at 298 K is 0.79 g-cm "3. What is the molar mass of the protein Assume that the density of the solution is the same as that of pure ethanol. See Exercise 8.95. 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

Corrinoid iron-sulfur protein (CFeSP) purified and characterized acetyl-CoA synthesis reconstituted from CH3-H4 folate, CO, and CoA using purified proteins. ... 

Mossbauer spectroscopy has been used to characterize the iron clusters in fuscoredoxin isolated from D. desulfuricans (133). The authors explained why the iron nuclearity was incorrectly determined, and studied the protein in three different oxidation states fully oxidized, one-electron reduced, and two-electron reduced. The error made in determining the iron cluster nuclearity was caused by the assumption that in the as-purified fuscoredoxin, cluster 2 is in a pure S = state. This assumption was proven to be false and unnecessary. In fact, the observation of four resolved, equal intensity (8% of total Fe absorption) spectral components associated with the S = i species in the as-purified protein is consistent with cluster 2 being a tetranuclear Fe cluster. The 4x8 = 32% Fe absorption for the four components indicates that only 64% of clusters 2 are in the S = state (the total Fe absorption for cluster 2 is 50% of the total Fe absorption). The remaining clusters 2 are in a different oxidation state, the spectrum of which is unresolved from that of cluster 1. 

The third purification procedure is based on the combination of temperature-triggered aggregation and affinity capture and has been used to not only purify proteins [50-54] but also other molecules [55, 56], and for the removal of pollutants from a solution [57-59]. For this procedure, ELP is conjugated to a capture reagent, which can be done either genetically or chemically. This procedure eliminates the need for cleavage of ELP after purification and introduces the potential to recycle the ELP. 

Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function. Investigations of purified proteins, eg, the Ca " ATPase of the sarcoplasmic reticulum, have in certain cases suggested that only a single protein and a single lipid are required to reconstitute an ion pump. 

Because of the risks of adverse reaction to the vaccine by persons who had already been exposed to the disease a sensitivity test must be carried out prior to immunization with BCG. A Mantoux skin test assesses an individual s sensitivity to a purified protein derivative (PPD) prepared fi om heat-treated antigens (tuberculin) extracted fiom M tuberculosis. A positive test imphes past infection or past, successful immunization Those with strongly positive tests may have active disease and should be referred to a chest clinic. Many people with active TB, especially disseminated TB, however, sero-convert fiom skin test positive to skin test negative. Results of the skin test must therefore be interpreted with care. 

Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures. 

PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with O.IM NaOH (data not shown). 

We described here the characterisation of the pemB gene and its product the second PME of E. chrysanthemi. The biochemical analysis of the purified protein indicated that PemB is actually an enzyme that demethylates pectin, leading to formation of methanol and PGA. However, PemB is more active on methylated oligogalacturonides than on polymeric pectin. The activating effect of non-ionic detergents on PemB was never pointed out for other pectinases and it is a characteristic of many membrane enzymes (21). 

Polyclonal Antibodies against FORL r purified polygalacturonase were raised in white rabbits. For the first immunization 200 jxg of purified protein in 300 jxl of distilled water was mixed with 200 1 of PBS and 500 n of complete Freund s adjuvcmt and injected intramusculary into the leg. Two subsequent intramuscular injections, each containing 300 fig of protein in 1 ml of incomplete Freund s adjuvant were given at 1 month intervals. Finally, the rabbit was bled 1 week later. The antisera, separated from blood by incubation at 37 "C, were stored in 1 ml fractions at -20 C. 

The function of the B domain has been confirmed by subcloning and preliminary kinetic measurements. We subcloned the AB domain of E. coli II, residues 348-637, after inserting a restriction site at a position corresponding to residue 348. The purified protein restored mannitol phosphorylation activity when measured with the A domain assay in Fig. 4A, and the B domain assay in Fig. 4B [42]. The B domain... 

In preliminary experiments the laboratory of Jockusch and coworkers in collaboration with Al-Awqati [32] has used indanyloxyacetic acid (IAA-94, Fig. 2, cf. below) to purify proteins between 30 and 150 kDa, which appear to be responsible for CP-transport and seem to be absent in one form of hereditary myotonia [8]. During the process of editing this review the Jentsch group has succeeded to clone the skeletal muscle CP-channel and yet another CP-channel [125,127]. 

The purified L-type channels from skeletal muscle appear to be a multisubunit protein containing the products of four different and unrelated genes. A cartoon depicting some of the features of the purified protein is shown in Fig. 1. The subunits of the channel are commonly referred to as the ai, a2, ]8, y, and 8. These have been observed to migrate in a variety of SDS-polyacrylamide gels with apparent... 

---