title: Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP.
abstract: The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) 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 β2M, 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 β2M that are suitable for further studies.
authors.affiliation: Department of Molecular Genetics, Institute of Experimental Medicine, Nord-West Branch of Russian Academy of Medical Sciences, Saint Petersburg, Russia. kaknikak@gmail.com
authors.forename: K V
authors.initials: KV
authors.lastname: Solovyov
authors.forename: D S
authors.initials: DS
authors.lastname: Polyakov
authors.forename: N A
authors.initials: NA
authors.lastname: Grudinina
authors.forename: V V
authors.initials: VV
authors.lastname: Egorov
authors.forename: I V
authors.initials: IV
authors.lastname: Morozova
authors.forename: T D
authors.initials: TD
authors.lastname: Aleynikova
authors.forename: M M
authors.initials: MM
authors.lastname: Shavlovsky
mesh_terms: D000682:Amyloid / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D011228:Prealbumin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000682:Amyloid; D011228:Prealbumin; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; D049452:Green Fluorescent Proteins