RESUMO
Molecular interaction analysis is an essential technique for the study of biomolecular functions and the development of new drugs. Most current methods generally require manipulation to immobilize or label molecules, and require advance identification of at least one of the two molecules in the reaction. In this study, we succeeded in detecting the interaction of low-molecular-weight (LMW) compounds with a membrane protein mixture derived from cultured cells expressing target membrane proteins by using the size exclusion chromatography-mass spectrometry (SEC-MS) method under the condition of 0.001% lauryl maltose neopentyl glycol as detergent and atmospheric pressure chemical ionization. This method allowed us to analyze the interaction of a mixture of medicinal herbal ingredients with a mixture of membrane proteins to identify the two interacting ingredients. As it does not require specialized equipment (e.g., a two-dimensional liquid chromatography system), this SEC-MS method enables the analysis of interactions between LMW compounds and relatively high-expressed membrane proteins without immobilization or derivatization of the molecules.
Assuntos
Proteínas de Membrana , Cromatografia em Gel , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peso MolecularRESUMO
Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors (GPCRs). Here, we report the crystal structure of human prostaglandin (PG) E receptor subtype EP3 bound to endogenous ligand PGE2 at 2.90 Å resolution. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands.
Assuntos
Receptores de Prostaglandina E Subtipo EP3/agonistas , Receptores de Prostaglandina E Subtipo EP3/química , Sítios de Ligação , Cristalografia por Raios X , Dinoprostona/química , Dinoprostona/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.
Assuntos
Receptores de Prostaglandina E Subtipo EP4/química , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Regulação Alostérica , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Caprilatos/química , Caprilatos/metabolismo , Cristalografia por Raios X , Epoprostenol/análogos & derivados , Epoprostenol/química , Epoprostenol/metabolismo , Humanos , Ligantes , Bicamadas Lipídicas , Simulação de Acoplamento Molecular , Naftalenos/química , Naftalenos/metabolismo , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Fenilbutiratos/química , Fenilbutiratos/metabolismo , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Spodoptera/genéticaRESUMO
Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.
Assuntos
Antagonistas Muscarínicos/metabolismo , Pirenzepina/análogos & derivados , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Antagonistas Muscarínicos/química , Mutação , N-Metilescopolamina/química , N-Metilescopolamina/metabolismo , Pirenzepina/química , Pirenzepina/metabolismo , Receptor Muscarínico M2/antagonistas & inibidoresRESUMO
The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação Proteica , Proteínas/genéticaRESUMO
Recent advancements in structural biology have facilitated the elucidation of complexes involving G protein-coupled receptors (GPCRs) and their associated signal transducers, including G proteins and arrestins. A comprehensive analysis of these structures provides profound insights into the dynamics of signaling mechanisms. These structural revelations can potentially guide the development of drugs to minimize side effects through targeted and selective signaling. Understanding the binding modes of different signal-selective ligands is imperative for future drug research and development. Here, we conduct a comparative examination of the structural details of various GPCR-signal transducer complexes and delve into the molecular basis of the currently proposed signal selectivity.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ligantes , BiologiaRESUMO
Orexin 2 receptor (OX2R) is a G protein-coupled receptor (GPCR) whose activation is crucial to regulation of the sleep-wake cycle. Recently, inactive and active state structures were determined from X-ray crystallography and cryo-electron microscopy single particle analysis, and the activation mechanisms have been discussed based on these static data. GPCRs have multiscale intermediate states during activation, and insights into these dynamics and intermediate states may aid the precise control of intracellular signaling by ligands in drug discovery. Molecular dynamics (MD) simulations are used to investigate dynamics induced in response to thermal perturbations, such as structural fluctuations of main and side chains. In this study, we proposed collective motions of the TM domain during activation by performing 30 independent microsecond-scale MD simulations for various OX2R systems and applying relaxation mode analysis. The analysis results suggested that TM3 had a vertical structural movement relative to the membrane surface during activation. In addition, we extracted three characteristic amino acid residues on TM3, i.e., Q1343.32, V1423.40, and R1523.50, which exhibited large conformational fluctuations. We quantitatively evaluated the changes in their equilibrium during activation in relation to the movement of TM3. We also discuss the regulation of ligand binding recognition and intracellular signal selectivity by changes in the equilibrium of Q1343.32 and R1523.50, respectively, according to MD simulations and GPCR database. Additionally, the OX2R-Gi signaling complex is stabilized in the conformation resembling a non-canonical (NC) state, which was previously proposed as an intermediate state during activation of neurotensin 1 receptor. Insights into the dynamics and intermediate states during activation gained from this study may be useful for developing biased agonists for OX2R.
Assuntos
Simulação de Dinâmica Molecular , Receptores de Orexina , Receptores de Orexina/química , Receptores de Orexina/metabolismo , Transdução de Sinais , HumanosRESUMO
Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.
Assuntos
Metionina , Humanos , Orexinas , Ligantes , Receptores de Orexina/genética , Receptores de Orexina/química , Espectroscopia de Ressonância MagnéticaRESUMO
Orexin receptors are a family of G protein-coupled receptors that consist of two subtypes: orexin-1 receptors (OX1Rs) and OX2Rs. They are expressed throughout the central nervous system and are involved in regulating the sleep-wake cycle. The development of antagonists to orexin receptors has become important in drug discovery because modulation of these receptors can lead to novel treatments for diseases related to the regulation of sleep and wakefulness, such as insomnia. In this study, we determined that the structure of OX2R bound to lemborexant, a dual orexin receptor antagonist (DORA), at 2.89 Å resolution. Comparisons of kinetic and dynamic properties of DORAs based on structures and simulations suggest that the enthalpy of molecular binding to receptors and the entropy derived from intramolecular structure can be separately controlled. These results complement existing structural information and allow us to discuss the usefulness of pharmacophore models and target selectivity to OXRs.
Assuntos
Desenho de Fármacos , Piridinas , Orexinas , Receptores de Orexina/genéticaRESUMO
Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.
Assuntos
Dinoprostona , Receptores de Prostaglandina E , Aminoácidos , Microscopia Crioeletrônica , Dinoprostona/farmacologia , Humanos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismoRESUMO
The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm-1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.
RESUMO
The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.
Assuntos
Receptor Muscarínico M2/química , Humanos , Ligantes , Análise EspectralRESUMO
Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution of 3.3 Å. In this structure, compared with that in the inactive EP4 structure, the sixth transmembrane domain is shifted outward on the intracellular side, although the shift is smaller than that in other class A GPCRs bound to Gs. Instead, the C-terminal helix of Gs is inserted toward TM2 of EP4, and the conserved C-terminal hook structure formsthe extended state. These structural features are formed by the conserved residues in prostanoid receptors (Phe542.39 and Trp3277.51). These findings may be important for the thorough understanding of the G protein-binding mechanism of EP4 and other prostanoid receptors.
Assuntos
Proteínas de Ligação ao GTP/química , Receptores de Prostaglandina E Subtipo EP4/química , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células Sf9 , SpodopteraRESUMO
The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-A (1 A=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic alpha-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Bactérias/genética , Caseínas , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/genética , Modelos Moleculares , Chaperonas Moleculares , Mutação , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Thermus thermophilus/metabolismoRESUMO
M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine and mediates various cellular responses in the nervous system. Here, we used attenuated total reflection-Fourier transform infrared spectroscopy analyses on M2R reconstituted in a lipid membrane to understand the molecular mechanism behind the ligand binding-induced conformational changes. Upon agonist binding, M2R shows large spectral change of the amide-I band corresponding to backbone CâO stretch, which likely connects with the receptor activation in the lipid environment. These results pave the way to probe effects of different ligand binding on GPCRs using vibrational spectroscopy.
Assuntos
Receptor Muscarínico M2/química , Sítios de Ligação , Humanos , Ligantes , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43â¯kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure Kd with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the Kd of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.
RESUMO
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.
Assuntos
Aminopiridinas/química , Azepinas/química , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Orexinas/química , Sulfonamidas/química , Triazóis/química , Aminopiridinas/metabolismo , Animais , Azepinas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia/métodos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Orexina/metabolismo , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/metabolismo , Síncrotrons , Termodinâmica , Triazóis/metabolismoRESUMO
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.