RESUMO
For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.
Assuntos
Clonagem Molecular , Oryza/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Escherichia coli/genética , Vetores Genéticos , Oryza/química , Proteínas de Plantas/química , Proteínas Recombinantes de Fusão/química , SolubilidadeAssuntos
Ressonância Magnética Nuclear Biomolecular , Oryza/química , Células Fotorreceptoras/química , Fitocromo/análise , Fitocromo/química , Fatores de Transcrição/análise , Fatores de Transcrição/química , Sequência de Aminoácidos , Aminoácidos Aromáticos/química , Isótopos de Carbono , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Isótopos de Nitrogênio , Fitocromo/isolamento & purificação , Fitocromo B , Estrutura Terciária de Proteína , Prótons , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Many seed storage proteins, including monomeric 2S albumin and polymeric prolamin, contain conserved sequences in three separate regions, termed A, B, and C, which contain the consensus motifs LxxC, CCxQL, and PxxC, respectively. Protein-sorting mechanisms in rice (Oryza sativa) endosperm were studied with a green fluorescent protein (GFP) fused to different segments of rice alpha-globulin, a monomeric, ABC-containing storage protein. The whole ABC region together with GFP was efficiently transported to protein storage vacuoles (type II protein bodies [PB-II]) in the endosperm cells and sequestered in the matrix that surrounds the crystalloids. Peptide Gln-23 to Ser-43 in the A region was sufficient to guide GFP to PB-II. However, GFP fused with the AB or B region accumulated in prolamin protein bodies. Substitution mutations in the CCxQL motif in the B region significantly altered protein localization in the endosperm cells. Furthermore, protein extracts containing these substituted proteins had increased amounts of the endoplasmic reticulum (ER) chaperons BiP (for binding protein), protein disulfide isomerase, and calnexin as a part of protein complexes that were insoluble in a detergent buffer. These results suggest that the ER chaperons and disulfide bonds formed at the dicysteine residues in CCxQL play critical roles in sorting fused proteins in the endosperm cells.
Assuntos
Dissulfetos/metabolismo , Oryza/embriologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , alfa-Globulinas/química , alfa-Globulinas/metabolismo , Sítios de Ligação/fisiologia , Sequência Conservada/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Chaperonas Moleculares/metabolismo , Oryza/genética , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Prolaminas , Ligação Proteica/fisiologia , Dobramento de Proteína , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sementes/genética , Sementes/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon. Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.
