RESUMO
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Assuntos
Fracionamento Químico/métodos , Físico-Química/métodos , Engenharia de Proteínas/métodos , Proteômica/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
PAHdb, a legacy of and resource in genetics, is a relational locus-specific database (http://www.pahdb.mcgill.ca). It records and annotates both pathogenic alleles (n = 439, putative disease-causing) and benign alleles (n = 41, putative untranslated polymorphisms) at the human phenylalanine hydroxylase locus (symbol PAH). Human alleles named by nucleotide number (systematic names) and their trivial names receive unique identifier numbers. The annotated gDNA sequence for PAH is typical for mammalian genes. An annotated gDNA sequence is numbered so that cDNA and gDNA sites are interconvertable. A site map for PAHdb leads to a large array of secondary data (attributes): source of the allele (submitter, publication, or population); polymorphic haplotype background; and effect of the allele as predicted by molecular modeling on the phenylalanine hydroxylase enzyme (EC 1.14.16.1) or by in vitro expression analysis. The majority (63%) of the putative pathogenic PAH alleles are point mutations causing missense in translation of which few have a primary effect on PAH enzyme kinetics. Most apparently have a secondary effect on its function through misfolding, aggregation, and intracellular degradation of the protein. Some point mutations create new splice sites. A subset of primary PAH mutations that are tetrahydrobiopterin-responsive is highlighted on a Curators' Page. A clinical module describes the corresponding human clinical disorders (hyperphenylalaninemia [HPA] and phenylketonuria [PKU]), their inheritance, and their treatment. PAHdb contains data on the mouse gene (Pah) and on four orthologous mutant mouse models and their use (for example, in research on oral treatment of PKU with the enzyme phenylalanine ammonia lyase [EC 4.3.1.5]).