EXPRESSION INE. coliAND 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.

Bio-specific immobilization of enzymes on electrospun PHB nanofibers

“…1A, lower panel). It has been reported that the functionality of a uorescence protein can be enhanced when fused with a foreign protein sequence [17,18]. However, the enzymatic activity of LipM7 exhibited greater dependence on the location of phasin fusion.…”

Polylactic Acid Nanoparticles Influence on Immunogenicity of the Protein Bound With Them

“…В нашем распоряжении имелась плазмида pTRC99a_P7 (Amersham-Pharmacia), кодирующая sfGFP (green fluorescent protein «superfolder»), помещенный под бактериальным промотором. Кроме того, в отделе молекулярной генетики ФГБНУ ИЭМ были созданы экспрессионные генетические конструкции для синтеза в прокариотической системе белка слияния β 2 M-sfGFP [13]. Для по-лучения белков использовали экспрессионный штамм E. coli BL21(DE3).…”

“…В качестве модели для выяснения влияния наночастиц из полимолочной кислоты на им-муногенность связанного с ними белка был выбран созданный нами ранее [12,13] реком-бинантный белок слияния β 2 -микроглобулина человека и зеленого флуоресцентного белка (β 2 M-sfGFP). Преимущество данного белка со-стоит в том, что он, во-первых, синтезируется и выделяется при помощи доступных стандарт-ных методов.…”

“…1). Ре-комбинантный белок получали из клеток E. coli штамма BL21(DE3), трансформированных экс-прессионной конструкцией pTRCb2msf [12,13]. В результате для данной работы было синтези-ровано и очищено 23 мг целевого белка из 3 л бактериальной культуры.…”

Extending the spatiotemporal resolution of super-resolution microscopies using photomodulatable fluorescent proteins

“…It has been reported that FPs with super folding abilities, such as sfGFP,°u oresce even if their fusion protein is expressed in inclusion bodies. 36 Thus, one can use this method to rapidly determine whether an FP has good folding and maturation properties. …”