ABSTRACT
Detailed biophysical studies of integral membrane proteins are often hampered by sample preparation difficulties. Membrane proteins are typically difficult to express in sufficient amounts to enable the use of demanding techniques such as nuclear magnetic resonance and X-ray crystallography for structural biology. Here, we show that an inexpensive batch-based cell-free expression system can be a viable alternative for production of a wide range of different membrane proteins, both of prokaryotic and eukaryotic origin. Out of 38 tested protein constructs, 37 express at levels suitable for structural biology, i.e. enough to produce several milligrams of protein routinely and without excessive costs. This success rate was not anticipated and is even more impressive considering that more than half of the expressed proteins where of mammalian origin. A detergent screen identified Brij-58 as the, in general, most successful choice for co-translational solubilization of the expressed proteins.
Subject(s)
Cell-Free System/metabolism , Cloning, Molecular/methods , Escherichia coli/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Animals , Cetomacrogol/chemistry , Circular Dichroism , Escherichia coli/metabolism , Gene Expression , Humans , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Protein Biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SolubilityABSTRACT
Experimental design principles were applied on cell-free protein synthesis to optimize performance with regard to the expression yield and the incorporation efficiency of amino acid precursors. A versatile screening platform based on batch-mode cell-free expression and central composite design was used. The performance of different extracts (S12 and S30), the concentration dependence of key components and the effect of different additives were investigated. We find that the initial expression yield can be enhanced twofold to threefold in this manner. The improved conditions comprise a modified S12 extract, optimized concentrations of creatine phosphate and key amino acids, as well as introduction of ketoacid additives. Our results show that current cell-free expression technology is far from optimal and that higher yields and increased utilization of the provided precursors are attainable with further optimization.
Subject(s)
Cell-Free System , Protein Biosynthesis , Amino Acids/metabolism , Escherichia coli/genetics , Escherichia coli/metabolismABSTRACT
BACKGROUND: The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. RESULTS: We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. CONCLUSION: This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.
Subject(s)
Evolution, Molecular , Petromyzon/genetics , Receptor, Melanocortin, Type 1/genetics , Receptor, Melanocortin, Type 4/genetics , Adrenocorticotropic Hormone/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Cell Line, Transformed , Cosyntropin/metabolism , Cyclic AMP/metabolism , Gene Duplication , Gene Library , Hagfishes/genetics , Humans , Molecular Sequence Data , Organ Specificity , Peptide Fragments/metabolism , Peptides, Cyclic/metabolism , Phylogeny , Pro-Opiomelanocortin/genetics , Protein Binding , Protein Interaction Mapping , Receptors, Melanocortin/metabolism , Recombinant Fusion Proteins/metabolism , Second Messenger Systems , Sequence Alignment , Sequence Homology, Amino Acid , Skin/metabolism , Species Specificity , Viscera/metabolism , alpha-MSH/analogs & derivatives , alpha-MSH/metabolism , beta-MSH/metabolism , gamma-MSH/metabolismABSTRACT
The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.
