RESUMO
The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and ß2-microglobulin (ß2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and ß2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and ß2M that are suitable for further studies.
Assuntos
Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Pré-Albumina/genética , Proteínas Recombinantes de Fusão/genética , Microglobulina beta-2/genética , Amiloide/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/isolamento & purificação , Humanos , Pré-Albumina/química , Pré-Albumina/isolamento & purificação , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Microglobulina beta-2/química , Microglobulina beta-2/isolamento & purificaçãoAssuntos
Amiloide/química , Lactalbumina/química , Peptídeos/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Simulação por Computador , Humanos , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Symmetrical peptide GYDTQAIVENNESTEYG (WT, Wild Type) identical to 35-51 aminoacid residues of human alpha-lactalbumin (HLA) and peptide GYDTQTVVNNNGHTDYG (ID, IDeal symmetry) homologous to beta-domain of mammalian alpha-lactalbumins can form amyloid-like fibrils in conditions required for fibrillogenesis of HLA. The latter peptide can also form fibrils in deionized water. Fibrils formed by these peptides can cause forming of HLA amyloid-like aggregates in physiological conditions. These results provide an evidence for presence of amyloidogenic determinant in beta-domain of alpha-lactalbumin. Thus, symmetry in the primary structure may play the role in fibrillogenesis of proteins.
Assuntos
Lactalbumina/química , Sequência de Aminoácidos , Amiloide/biossíntese , Humanos , Lactalbumina/genética , Microscopia de Força Atômica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de SequênciaRESUMO
Polypeptide chain fragments of recombinant transthyretin (TTR) with leucine-55 substituted by proline (L55P), which are involved in abnormal fibrillogenesis of this protein, were studied. No fibrils were produced in purified preparations of TTR(L55P) under the optimum conditions for fibrillogenesis but in absence of protease inhibitors. The ability of TTR for fibrillogenesis was lost because of a limited proteolysis resulting in detachment of the TTR polypeptide chain C-terminal fragment of approximately 18 amino acid residues in length. This proteolysis seemed to occur with involvement of a bacterial serine endopeptidase sohB (EC 3.4.21), which was identified in TTR preparations by the MALDI-TOF method. The presence of the C-terminal fragment of the TTR polypeptide chain seems to be crucial for production of abnormal fibrils.
