Nonspecific recombination

Ab, B.K., Kiessling, R., Van, E.J.D., Thole, J.E.. Kumararatne, D.S., Pisa, P., Wondimu. A., Ottenhoff, T.H. (1990). Induction of antigen-specific CD4+ HLA-DR restricted cytotoxic lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial 65 kDa heat shock protein. Eur. J. Immunol. 20, 369-377. 

By using an in vitro UV-crosslinking approach, bacteria-purified recombinant CPEB3 has been demonstrated to interact with the 2> UTR of GluR2 mRNA. To test whether this interaction occurs in vivo, hippocampal neuron cultures are ultraviolet (UV)-irradiated, homogenized, and immunoprecipitated with CPEB3 IgG or nonspecific IgG (Huang et al, 2006). Three plates of 2- to 3-week-old cultured neurons ( 6—8 million... 

Supramolecular chain scission differs further from covalent chain scission, because supramolecular recombination is typically the predominant fate of a ruptured chain anthropomorphically speaking, the supramolecular moieties are, by their very nature, predisposed to reassociation rather than alternative reaction pathways. This predisposition is not intrinsic to the products of covalent bond mpture, which might lead either to high-energy intermediates with nonspecific reactivity or to species that require catalyst or elevated temperature to recombine. The... 

Fig. 4. Schematic of a single-step array fabrication process for in vivo biotinylated proteins. Step a A cmde lysate containing the desired biotinylated recombinant protein is printed onto a streptavidin-coated surface coderivatized with a polymer that resists nonspecific protein absorption. Step b Unbound proteins are washed away to leave the purified recombinant protein, specifically immobilized and oriented on the array surface via the biotin moiety on the BCCP tag.

The unique benefit of recombinant microbes for screening is that target specificity is evaluated early in the process. Compounds that nonspecifically affect enzyme function (by sulfhydryl modification, cation chelation, alkylation, etc.) will not survive the comparative testing phase. The hit rate in our laboratories in these types of screens has been under 0.01% (unpublished observations), which means that additional attention can be focused only on the most promising candidates. In contrast, screens that employ purified recombinant enzymes for HTS often have hit rates >2.5% (unpublished observations). Further characterization of so many candidates requires a considerable effort in both biology and chemistry laboratories. 

M. D. Jones, L. A. Merewether, C. L. Clogston, and H. S. Lu, Peptide map analysis of recombinant human granulocyte colony stimulating factor elimination of methionine modification and nonspecific cleavages, Anal. Biochem., 276 135 (1994). 

As in the case of chemical inhibition, a lack of specificity can complicate the interpretation of antibody inhibition experiments. A lack of specificity and the nonspecific effects outlined above likely account for the majority of cases where the sum of the inhibitory effects of a panel of inhibitory antibodies adds up to greater than 100%. Another potential problem stems from the fact that many antibodies do not completely inhibit the activity of a microsomal CYP enzyme or the corresponding recombinant CYP enzyme. If an antibody inhibits the metabolism of a marker substrate by 80%, and if the same antibody inhibits the metabolism of drug candidate by 80%, there is uncertainty as to whether the inhibited enzyme contributes 80% or 100% to the metabolism of the drug candidate. This may seem like a trivial difference, but it has a... 

Certain circumstances might arise where the bioanalyst wants to examine a recombinant protein preparation for low level protein impurities but a suitable antiserum is unavailable and the variability and nonspecificity of silver staining is unacceptable. An additional assay would be a Western blot analysis after derivatization of the protein mixture with Sanger s reagent... 

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