Protein alternatives

Figure 7.2 Schematic showing the relationship of the native antigen to the peptide mimic. The native antigen (a protein) is shown as a winding, twisted line, so as to represent a hypothetical three-dimensional structure. The peptide represents the antibody-binding epitope (shown in dotted lines) of the native antigen. The epitope can represent a linear sequence of the native protein. Alternatively, the epitope can be formed by amino acids that are not immediately adjacent to each other in the primary sequence but brought together by the three-dimensional folding of the protein. Adapted with permission from Sompuram et al.6...

Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.5-0.1 M EDC concentration in the reaction mixture usually works well. To make it easier to add the correct quantity of EDC, a higher concentration stock solution may be prepared if it is dissolved and used immediately. To prepare the peptide-protein conjugate, add the solution from step 3 to 10 mg of EDC in a test tube. Mix to dissolve. If this ratio of EDC to peptide or protein results in precipitation, scale back the amount of carbodiimide addition until a soluble conjugate is obtained. For some proteins, as little as 0.1 times this amount of EDC may have to be used to maintain solubility. 

Reduce disulfides in the protein sample by the addition of 2 pi of 50 mM TCEP (Thermo Fisher) to each 100 pi aliquot of protein solution (final concentration 1 mM). Cover and boil the samples for 10 minutes in a water bath to completely denature and reduce the proteins. Alternatively, reduction may be done at 60°C for 1 hour. Avoid the use of thiol-containing reductants, such as DTT, as these will react with the thiol blocking agent used in the next step. 

Figure 10.4. Translation of the genetic blueprint into proteins. Alternative splicing leads to isoforms. After translation proteins are often modified to become functional.

A recently discovered subset of triple-stranded /l-helices from bacteriophage tail proteins (alternatively termed triple-stranded /1-solenoids ) represents another distinct group of /1-fibrous folds (Fig. 3B). In these structures, three identical chains related by threefold rotational symmetry wind around a common axis. These chains form unusual parallel /1-sheets with no intra- and only intermolecular -structural hydrogen bonding. Kajava and Steven (this volume) survey the distinguishing structural features of the known triple-stranded /1-solenoids, also documenting their notable diversity and differences in comparison to the single-stranded /1-solenoids. 

The unique size, polarizability, and geomertric properties of the C-Se anion may ultimately be exploited toward zinc complexation in a selenocysteine-engineered protein alternatively, this group might be incorporated into a potent zinc-enzyme inhibitor. 

Figure 7.19 Proposed metabolic activation of paracetamol to a toxic, reactive intermediate /V-acetyl-p-benzoquinone imine (NAPQI). This can react with glutathione (GSH) to form a conjugate or with tissue proteins. Alternatively, NAPQI can be reduced back to paracetamol by glutathione, forming oxidized glutathione (GSSG).

The aerobic pathway of metabolism (pathway 1) (Fig. 7.77) produces trifluoroacetyl chloride, a highly reactive acyl chloride, which can react with nucleophiles such as amino groups similar to those on proteins. Alternatively, reaction with water yields trifluoroacetic acid. Trifluoroacetylchloride is the probable reactive metabolite that trifluoroacylates protein, most probably at lysine residues (Fig. 7.77). Removal of the trifluoroacyl moiety from the... 

There are two approaches to isolating a membrane-associated protein. In one method, the relevant membrane fraction can first be prepared and then used to isolate the protein. Alternatively, whole tissue can be subjected to an extraction that solubilizes the membranes and releases the cytoplasmic contents as well. The former is much better in that purification is accomplished by isolating the membranes the specific activity of the solubilized membrane fraction will be much higher than in the second method. However, the process of purifying the membrane fraction may lead to substantial losses, and it may be difficult to scale up. If total recovery of the protein is more important than purity, a whole-tissue extract is likely to be more appropriate. Although this... 

Diafiltration is a variation of ultrafiltration, in which fresh solvent is added to the feed solution to replenish the volume ultrafiltered, and in the process washes small molecules such as salts away from the retained macromolecules. Using appropriate replenishing solutions, diafiltration is a common procedure to perform buffer exchange of proteins. Alternatively, a dilute solution may be first ultrafiltered to concentrate the feed material, then diafiltered to purify the retentate. It is sometimes possible to fractionate a mixture of macrosolutes by sequential diafiltration with a series of membranes of progressively lower molecular weight cutoff ratings. 

DOR-1 encodes both delta and delta2 receptor subtypes. Therefore, the different properties of the delta and delta2 receptor subtypes could result from differential posttranslational modification of the DOR-1-encoded receptor protein, alterations in the molecular environment of the receptor protein. Alternatively, the understanding of the existed splice variants for DOR-1 must be important to accept the concept of the deltai and delta2 receptor subtypes, as suggested by DOR-1 antisense mapping studies [60]. 

This pictorial representation of a possible mechanism of action of a polar ion-pairing reagent has allowed prediction of what anions will be useful in decreasing the retention of a given protein. Alternatively, a nonpolar anion such as hexane sulfonate could be expected to associate with an ammonium ion present in the sample, with an increase in the nonpolar surface area and hence retention time. For example, apolipopro-tein C-III, which is readily eluted by an organic solvent gradient from a Cig column in the presence of phosphate anions, is retained indefinitely if butanesulfonate is used as the counterion. 

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