RESUMO
Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a ~ 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Clonagem Molecular/métodos , Chaperoninas do Grupo I/genética , Chaperonas Moleculares/genética , Proteínas de Ligação a Fosfato/genética , Ribulose-Bifosfato Carboxilase/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Chaperoninas do Grupo I/metabolismo , Histidina/genética , Histidina/metabolismo , Cinética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fotossíntese/genética , Dobramento de Proteína , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ribulose-Bifosfato Carboxilase/isolamento & purificação , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes.
Assuntos
Espectrometria de Massas/métodos , Redes e Vias Metabólicas , Mapeamento de Interação de Proteínas/métodos , Proteína Fosfatase 2/química , Chaperoninas/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Humanos , Conformação ProteicaRESUMO
Hsp70 has high potential as an immune-adjuvant molecule: it mediates cytokine expression and maturation of antigen presenting cells (APCs) and also elicits a cytotoxic T-lymphocyte (CTL) response to antigenic peptides. How Hsp70 interacts with APCs is only poorly understood. Various surface proteins have been implicated in binding Hsp70 but their role in antigen presentation has remained controversial. The specific aim of this work was to determine the binding and uptake of human full-length Hsp70 as well as its separate ATPase (N70) and substrate-binding domains (C70) by APCs. Using laser scanning microscopy and FACS analysis, we established the existence of at least two distinct receptors for Hsp70, which are localized to distinct microdomains of the APC membrane. These receptors interact with the N70 and C70 domains of Hsp70, respectively. This observation was supported by the finding of a substantial portion of Hsp70 and C70, but not N70, in a detergent resistant membrane fraction. Accordingly, C70 and N70 did not compete with each other for binding. The bound proteins were rapidly internalized, with N70 and C70 localizing to separate endosomal compartments. Similarly, internalized free and peptide-loaded Hsp70 segregated rapidly within the cell. Efficient cross presentation of antigenic peptide bound to Hsp70 or C70 was demonstrated with the B3Z read out system. Consequently, the interaction of C70 with its putative receptor seems to be responsible for Hsp70-mediated cross presentation. Future studies should make use of C70 in identifying the uptake receptor of Hsp70-peptide complexes. In addition we could observe a stimulation of uptake of free peptide by preincubation with Hsp70 and N70, but not C70, whereas an Hsp-dependent cytokine secretion could not be detected. Consequently, by employing the individual domains it may be possible to distinguish between the different outcomes of Hsp70 treatment, like immune stimulation, DC maturation and antigen-specific responses.