No abstract

This report summarizes updated CDC guidance for the evaluation, management, and follow‐up of people with lung injury related to electronic cigarette or vaping product use.

No abstract

No abstract

No abstract

Current approaches to protein site-directed mutagenesis require an independent user operation for each mutation. This can impede large-scale scanning mutagenesis projects such as mapping protein interaction surfaces, active sites, or epitopes. It also prevents the creation of protein libraries of defined complexity for directed evolution purposes. Here we present a simple, fast, and effective way to perform scanning codon mutagenesis throughout a protein sequence. The process allows the researcher to define the new codon change therefore any amino acid mutation can be achieved. We demonstrate this approach by creating a library of proteins that contain single unnatural amino acid mutations encoded by the amber stop codon, TAG. The mutant proteins generated by this method can be expressed and assayed individually, or used together as a mixed population of "rationally diversified" protein sequences.Keywords scanning mutagenesis; amino acid; unnatural; transposon; photo-crosslinking Whether through chemical synthesis or genetic mutagenesis, the ability to rationally change the amino acid sequence of proteins has had a profound impact upon the understanding of structure and function. Diversity-oriented approaches to protein mutagenesis such as errorprone PCR(1), DNA shuffling(2), or cassette mutagenesis(3) have expanded on this and can easily create mixtures of complexity that exceed protein screening capabilities. Scanning mutagenesis utilizes a different approach by making small changes, such as single alanine replacements(4), so that the contribution of individual side chains can be deciphered and later exploited. This generates mixtures of modest, defined complexity and is well suited for mapping protein-protein interactions, solvent accessibility, or active sites using low to mediumthroughput assays. The limitation, however, is the time and effort spent creating the mutations via PCR-based methods. For example there are a clear, defined number of single alanine mutants that could be made of a protein 500 amino acids long, but generating those mutants with conventional methods is expensive, time consuming and most likely the limiting factor when considering such a project.We faced this conceptual challenge when seeking to investigate protein surfaces using photocrosslinking unnatural amino acids that are genetically encoded by the amber stop codon, TAG *correspondence: acropp@umd.edu. AUTHOR CONTRIBUTIONS KAD and TAC designed the research; KAD and ML performed the experiments; KAD and TAC analyzed the results: KAD, ML, and TAC wrote the paper.Supporting Information Available Construction and sequences of plasmids used in the study. This material is available free of charge via the Internet. Previously, transposon methods have been used to map protein domains via randomly distributed amino acid linker insertions (10-12). Recently Jones and co-workers described a transposon-based system for removal of triplet nucleotides from plasmid DNA using TypeIIS asymmetric restriction enzymes (13) and have gone on re...

No abstract

No abstract