RESUMO
Transient transfection of human embryonic kidney cells (HEK 293) enables the rapid and affordable lab-scale production of recombinant proteins. In this chapter protocols for the expression and purification of both secreted and intracellular proteins using transient expression in HEK 293 cells are described.
Assuntos
Escherichia coli/genética , Células HEK293/metabolismo , Mamíferos/genética , Proteínas Recombinantes/genética , Animais , Escherichia coli/metabolismo , Humanos , Mamíferos/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção/métodosRESUMO
In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.
Assuntos
Anticorpos Monoclonais/genética , Fragmentos Fab das Imunoglobulinas/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos/genética , Células HEK293 , Humanos , Hibridomas/citologia , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , TransfecçãoRESUMO
The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.
Assuntos
Proteínas/genética , Proteínas/metabolismo , Animais , Baculoviridae/genética , Células CHO , Células COS , Técnicas de Cultura de Células/métodos , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular/métodos , Cricetinae , Cricetulus , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , Células VeroRESUMO
The EphA4 tyrosine kinase cell surface receptor regulates an array of physiological processes and is the only currently known class A Eph receptor that binds both A and B class ephrins with high affinity. We have solved the crystal structure of the EphA4 ligand binding domain alone and in complex with (1) ephrinB2 and (2) ephrinA2. This set of structures shows that EphA4 has significant conformational plasticity in its ligand binding face. In vitro binding data demonstrate that it has a higher affinity for class A than class B ligands. Structural analyses, drawing on previously reported Eph receptor structures, show that EphA4 in isolation and in complex with ephrinA2 resembles other class A Eph receptors but on binding ephrinB2 assumes structural hallmarks of the class B Eph receptors. This interactive plasticity reveals EphA4 as a structural chameleon, able to adopt both A and B class Eph receptor conformations, and thus provides a molecular basis for EphA-type cross-class reactivity.
Assuntos
Efrinas/química , Efrinas/metabolismo , Receptor EphA4/química , Receptor EphA4/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, can be transfected with high efficiency (> 50% cell expression) and are amenable to culture at multi-litre scale. Growing cells in micro-plate format allows screening of large numbers of vectors in parallel to prioritise those amenable to scale-up and purification for subsequent structural or functional studies. The glycoform of the expressed protein can be modified by treating cell cultures with kifunensine which inhibits glycan processing during protein synthesis. This results in the production of a chemically homogeneous glycoprotein with short mannose-rich sugar chains attached to the protein backbone. If required, these can be readily removed by endoglycosidase treatment.
