Dataset Information

Protein coexpression using FMDV 2A: effect of "linker" residues.

ABSTRACT: Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A "cleavage" may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

SUBMITTER: Minskaia E

PROVIDER: S-EPMC3710640 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Protein coexpression using FMDV 2A: effect of "linker" residues.

Minskaia Ekaterina E Ryan Martin D MD

BioMed research international 20130612

Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A "cleavage" may, however, be compromised by both the use of shorter versions of F2A and the sequences (d ...[more]

PMID: 23878801

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Project description:Identification of the residues in protein-protein interaction sites has a significant impact in problems such as drug discovery. Motivated by the observation that the set of interface residues of a protein tend to be conserved even among remote structural homologs, we introduce PrISE, a family of local structural similarity-based computational methods for predicting protein-protein interface residues.We present a novel representation of the surface residues of a protein in the form of structural elements. Each structural element consists of a central residue and its surface neighbors. The PrISE family of interface prediction methods uses a representation of structural elements that captures the atomic composition and accessible surface area of the residues that make up each structural element. Each of the members of the PrISE methods identifies for each structural element in the query protein, a collection of similar structural elements in its repository of structural elements and weights them according to their similarity with the structural element of the query protein. PrISEL relies on the similarity between structural elements (i.e. local structural similarity). PrISEG relies on the similarity between protein surfaces (i.e. general structural similarity). PrISEC, combines local structural similarity and general structural similarity to predict interface residues. These predictors label the central residue of a structural element in a query protein as an interface residue if a weighted majority of the structural elements that are similar to it are interface residues, and as a non-interface residue otherwise. The results of our experiments using three representative benchmark datasets show that the PrISEC outperforms PrISEL and PrISEG; and that PrISEC is highly competitive with state-of-the-art structure-based methods for predicting protein-protein interface residues. Our comparison of PrISEC with PredUs, a recently developed method for predicting interface residues of a query protein based on the known interface residues of its (global) structural homologs, shows that performance superior or comparable to that of PredUs can be obtained using only local surface structural similarity. PrISEC is available as a Web server at http://prise.cs.iastate.edu/Local surface structural similarity based methods offer a simple, efficient, and effective approach to predict protein-protein interface residues.

Dataset Information

Protein coexpression using FMDV 2A: effect of "linker" residues.

Publications

Protein coexpression using FMDV 2A: effect of "linker" residues.

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