RESUMO
The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.
Assuntos
Escherichia coli/genética , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes , Sequência de Bases , Varredura Diferencial de Calorimetria , Clonagem Molecular , Primers do DNA/genética , Peixes/genética , Linguado/genética , Congelamento , Expressão Gênica , Genes Sintéticos , Glicoproteínas/biossíntese , Glicoproteínas/isolamento & purificação , Gelo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sequências de Repetição em TandemAssuntos
Flavoproteínas/química , NADH NADPH Oxirredutases/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Complexo Piruvato Desidrogenase , Proteínas Recombinantes/química , Acetiltransferases/química , Azotobacter vinelandii/enzimologia , Di-Hidrolipoamida Desidrogenase/química , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Dissulfetos/metabolismo , Escherichia coli/enzimologia , Flavoproteínas/biossíntese , Geobacillus stearothermophilus/enzimologia , Glutationa Redutase/química , Modelos Estruturais , NADH NADPH Oxirredutases/biossíntese , Mutação Puntual , Proteínas Recombinantes/biossínteseRESUMO
A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads. The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography. Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant. The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A. The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit.
Assuntos
Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/química , Cromatografia por Troca Iônica , Cristalização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Glutationa Transferase/genética , Plasmídeos , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.
Assuntos
Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Cristalização , Escherichia coli/genética , Glutationa Transferase/genética , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Difração de Raios XRESUMO
Anhydrolevuglandin E2 (AnLGE2) is closely related to prostaglandin E2 (PGE2) and has been found to inhibit the uterotonic activity of PGE2. The binding of PGE2 and its inhibition by AnLGE2 has been determined in rat uterine membrane fractions. AnLGE2 inhibited the binding of 3HPGE2 in a dose related fashion. 3HAnLGE2 also binds to rat uterine membrane fractions and its binding is inhibited by PGE2 in a dose related fashion. These data support previous physiological observations that AnLGE2 inhibits the actions of PGE2 by acting at the PGE2 receptor. Thus, AnLGE2 appears to be a specific inhibitor of PGE2 actions at its uterine receptors.
