RESUMO
This article describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production, how to maximize and scale up recombinant protein production, and how to purify recombinant proteins. © 2018 by John Wiley & Sons, Inc.
Assuntos
Proteínas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Animais , Baculoviridae/genética , Expressão Gênica , Técnicas Genéticas , Biossíntese de Proteínas , Células Sf9RESUMO
Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.
Assuntos
Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Animais , Transporte Biológico , Colesterol/metabolismo , Ativação Enzimática , Expressão Gênica , Células HEK293 , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/química , Fosfatidilcolina-Esterol O-Aciltransferase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9RESUMO
LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/ß hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.
Assuntos
Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/química , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Colesterol/genética , Cristalografia por Raios X , Humanos , Mutação , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.
Assuntos
Anticorpos Monoclonais/imunologia , Colesterol/sangue , Testes de Neutralização , Serina Endopeptidases/imunologia , Animais , Colesterol/imunologia , Cristalografia por Raios X , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/genética , Receptores de LDL/fisiologiaRESUMO
Proprotein convertase subtilisin kexin type 9 (PCSK9) has been shown to be involved in the regulation of extracellular levels of the low-density lipoprotien receptor (LDLR). Although PCSK9 is a subtilase, it has not been shown to degrade the LDLR, and its LDLR-lowering mechanism remains uncertain. Here we report the crystal structure of human PCSK9 at 2.3 A resolution. PCSK9 has subtilisin-like pro- and catalytic domains, and the stable interaction between these domains prevents access to PCSK9's catalytic site. The C-terminal domain of PCSK9 has a novel protein fold and may mediate protein-protein interactions. The structure of PCSK9 provides insight into its biochemical characteristics and biological function.
Assuntos
LDL-Colesterol/sangue , Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , LDL-Colesterol/antagonistas & inibidores , Cristalografia por Raios X , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Dobramento de Proteína , Mapeamento de Interação de ProteínasRESUMO
This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild-type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.
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Baculoviridae/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Clonagem Molecular/métodos , Vetores Genéticos/fisiologia , Cultura de Vírus/métodos , Animais , Baculoviridae/genética , Baculoviridae/fisiologia , Linhagem Celular/metabolismo , Linhagem Celular/virologia , DNA Complementar/genética , DNA Recombinante/genética , DNA Viral/genética , Vetores Genéticos/genética , Indicadores e Reagentes , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/crescimento & desenvolvimento , Nucleopoliedrovírus/fisiologia , Spodoptera/citologia , Spodoptera/virologia , Transfecção/métodos , Ensaio de Placa ViralRESUMO
This unit describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production , how to maximize and scale up recombinant protein production, and how to purify recombinant proteins.
Assuntos
Baculoviridae/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Proteínas Recombinantes/isolamento & purificação , Animais , Baculoviridae/fisiologia , Linhagem Celular/metabolismo , DNA Recombinante/genética , Expressão Gênica , Glutationa Transferase/análise , Histidina/análise , Indicadores e Reagentes , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Spodoptera/citologia , Spodoptera/virologia , Coloração e Rotulagem/métodosRESUMO
Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post-translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.