Protein structure analysis recombination

PKR is a NADPH-dependent monomeric enzyme of 34 to 35 kDa belonging to the aldo-and keto-reductase superfamily.The first isolation and characterization of a PKR cDNA was from G. max The G. max cDNA, and cDNAs from other species, have been used to confirm the PKR activity of the recombinant protein, and to produce larger protein amounts for structural analysis.Studies of the recombinant protein, and analysis of 35SCaMV PKR transgenic plants, have also shown that PKR is able to function with CHS proteins from nonlegume species that synthesize only the common 5-hydroxyflavonoids. 

Capillary electrophoresis has found use in the biotechnology industry for structural analysis of recombinant proteins. The high resolving power of CE for charged analytes makes it a powerful tool for the analysis of tryptic digests. Therefore, many of the techniques given here, such as the determination of thiols, carbohydrates, and amino acids, will be employed for this purpose. 

Analysis of the primary protein structure of the human 5-HT1B receptor reveals a putative site for palmitoylation, i.e., a cysteine residue located in the short carboxyl tail of the receptor (141). A recombinant c-myc epitope-tagged 5-HT1B receptor was expressed in Sf9 insect cells and palmitoylation of the receptor was demonstrated by metabolic labeling of the cells with [3H]palmitic acid. 

This technique gives information about the protein s primary structure, which may include its amino and/or carboxyl terminal groups (Edman, 1950). For recombinant DNA-derived proteins, this analysis serves to confirm the amino acid sequence predicted by the DNA sequence. The analysis can also be useful to determine the protein s homogeneity. 

By fc-mcans cluster analysis with Euclidean distance in the segmental Q-coordinates, we divided the structure ensemble of the MD unfolding trajectories into nine clusters [25]. The clustering was performed using all data obtained for the authentic and recombinant proteins, and the clusters were numbered in the order of the distance from the native structure. Figure 2.10(c)-(f) shows protein structures in four representative clusters (Clusters 1, 4, 5, and 9), in which Cluster 1 is almost identical to the native structure with all of the 17 Q-coordinates close to unity, whereas Cluster 9, which lost 84% of its native contacts, represents the unfolded state. 

Structure analysis of several proteases involved in blood coagulation and fibrinolysis reveals a diverse, sometimes repetitive, assembly of discrete protein modules (Fig. 9.4) [56]. While these modules represent independent structural units with individual folding pathways, their concerted action contributes to function and specificity in the final protein product. On the genetic level, these individual modules are encoded in separate exons. Over the course of modular protein evolution, new genes are created by duplication, deletion, and rearrangement of these exons. Mechanistically, the exon shuffling actually takes place in the intervening intron sequences (intronic recombination - for further details see [10]). 

MALDI-MS Analysis of Recombinant Adenoviral Proteins Isolated from RP-HPLC. Another approach in structural analysis of adenoviral proteins is to inject intact viruses onto RP-HPLC and collect fractions for further mass analysis. Compared with SDS-PAGE assay, RP-HPLC is... 

Tomasselli, A.G., Heinrickson, R.L., and Watenpaugh, K.D. (1991). Recombinant DNA technology and crystaUography a new alliance in unraveling protein structure-function relationships. In Purification and Analysis of Recombinant Proteins. R.Seetharam and S.K.Sharma, eds. (New York Marcel Dekker), pp. 285 315. 

In x-ray crystallographic analysis of COMb it has been noted that there is no pathway for the migration of CO between the buried binding site and solvent. Therefore, rapid rearrangements of the protein structures should be accompanied by recombination of CO. The transient absorption study on the relaxation process of photolysis product of COMb revealed that the recombination of the photodissociated CO was nonexponential. Fe-CO stretching band that appears in Raman spectra, is a good maker for this distal pocket environment. We pursued the time profile of this band to monitor the dynamics and found an intermediate species common to all the mutants. We will discuss the existence of the intermediate which correspond to "open form". 

---