ABSTRACT
Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.
Subject(s)
Cell-Free System , Protein Biosynthesis/genetics , Proteins/genetics , Cloning, Molecular , Escherichia coli/genetics , Eukaryota/genetics , Gene Expression , Genetic Vectors , Germ Cells , Proteins/isolation & purification , Triticum/geneticsABSTRACT
The Center for Eukaryotic Structural Genomics (CESG) is a "specialized" or "technology development" center supported by the Protein Structure Initiative (PSI). CESG's mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG's platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.
Subject(s)
Genomics/organization & administration , Proteins/chemistry , Animals , Crystallography, X-Ray , Genomics/methods , Humans , Multi-Institutional Systems/organization & administration , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Proteins/genetics , Proteomics/organization & administrationABSTRACT
We identified a functional domain (XlePABP2-TRP) of Xenopus laevis embryonic type II poly(A)-binding protein (XlePABP2). The NMR structure of XlePABP2-TRP revealed that the protein is a homodimer formed by the antiparallel association of beta-strands from the single RNA recognition motif (RRM) domain of each subunit. In each subunit of the homodimer, the canonical RNA recognition site is occluded by a polyproline motif. Upon poly(A) binding, XlePABP2-TRP undergoes a dimer-monomer transition that removes the polyproline motif from the RNA recognition site and allows it to be replaced by the adenosine nucleotides of poly(A). Our results provide high-resolution structural information concerning type II PABPs and an example of a single RRM domain protein that transitions from a homodimer to a monomer upon RNA binding. These findings advance our understanding of RRM domain regulation, poly(A) recognition, and are relevant to understanding how type II PABPs function in mRNA processing and human disease.
Subject(s)
Poly(A)-Binding Protein II/chemistry , Poly(A)-Binding Protein II/metabolism , RNA/metabolism , Xenopus Proteins/chemistry , Xenopus Proteins/metabolism , Animals , Binding Sites , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Poly A/metabolism , Protein Conformation , Protein Structure, Tertiary , RNA/chemistry , Structure-Activity Relationship , Xenopus/metabolismABSTRACT
The MADS domain protein AGL15 (AGAMOUS-Like 15) has been found to preferentially accumulate in angiosperm tissues derived from double fertilization (i.e. the embryo, suspensor, and endosperm) and in apomictic, somatic, and microspore embryos. Localization to the nuclei supports a role in gene regulation during this phase of the life cycle. To test whether AGL15 is involved in the promotion and maintenance of embryo identity, the embryogenic potential of transgenic plants that constitutively express AGL15 was assessed. Expression of AGL15 was found to enhance production of secondary embryos from cultured zygotic embryos, and constitutive expression led to long-term maintenance of development in this mode. Ectopic accumulation of AGL15 also promoted somatic embryo formation after germination from the shoot apical meristem of seedlings in culture. These results indicate that AGL15 is involved in support of development in an embryonic mode.
