Design and Gene Engineering Synthesis of an Extremely Thermostable Protein with Biological Activity

Abstract

A novel strategy for designing and synthesizing extremely thermostable biologically active proteins is proposed. The design concept is based on combining a rigid and extremely hydrophobic peptide unit with a biologically active peptide unit. The cell adhesive peptide sequence, Arg-Gly-Asp (RGD), as a functional peptide unit was incorporated into the elastin-based rigid polyhexapeptide, whose repeating unit is Ala-Pro-Gly-Val-Gly-Val (APGVGV). The designed fusion gene was expressed in E. coli, and the resulting protein, designated ER4, was purified with affinity chromatography. The ER4-coated cell culture plate showed sufficient cell adhesive activity through the RGD sequence on the surface of ER4. The thermostability of ER4 was demonstrated by estimating the remaining cell adhesive activity after autoclaving at 120 degrees C for 20 min, and it retained over 90% of cell adhesive activity compared with native ER4.

Hydrogel scaffolds composed of genetically synthesized self-assembling peptides for three-dimensional cell culture

“…[2][3][4][5] Artificial ECM proteins have been designed and genetically synthesized in our laboratory. [6][7][8][9][10] Our strategy for the design of artificial ECM proteins relies on the combination of structural peptides and active functional peptides that control cellular functions. One of the ECM proteins previously constructed in our lab, named ERE, is composed of 12 repeats of the elastin-derived APGVGV peptide motif as the stable structural peptide, with the cell adhesive RGD peptide motif as the active functional peptide.…”

“…7 Our genetically designed ECM proteins based on ERE demonstrated applicability as biomaterials. [6][7][8][9][10] However, covalent cross-linking is required to form a hydrogel structure in order to apply our designed ECM proteins to three-dimensional (3-D) culture. 13 The use of cross-linking reagents can result in the loss of the functional peptide's activity.…”

“…One of the ECM proteins previously constructed in our lab, named ERE, is composed of 12 repeats of the elastin-derived APGVGV peptide motif as the stable structural peptide, with the cell adhesive RGD peptide motif as the active functional peptide. 6,7 ERE can be immobilized on a hydrophobic surface via the hydrophobic APGVGV peptide motif and exhibits cell adhesive activities as a result of the RGD peptide. Multi-functional ECM proteins have also been constructed based on the ERE protein.…”

Construction of epidermal growth factor fusion protein with cell adhesive activity

“…This sequence is a motif found in elastin which provides a combination of strength and flexibility in the extracellular matrix. We designated the elastin motif as E and the RGD sequence as R. In a previous study we constructed a stable cell adhesive protein which we designated ER4, four repeats of the sequence elastin-RGD (ER) [29]. In the ER4 protein the elastin peptide (E) consisted of 12 repeats of the APGVGV sequence (E12), which has a hydrophobic rigid b-spiral structure, and RGD sequence with cell adhesive function.…”

Construction of a multi‐functional extracellular matrix protein that increases number of N1E‐115 neuroblast cells having neurites

“…One of those, called ER4, consisted of 4 repeats of the fusion construct that conjugated (APGVGV) 12 to the GRGDS sequence. It had been reported that ER4 retained cell adhesive activity due to the incorporated RGDS peptide, 19,20 and therefore ER4 was considered as a positive control for the other artificial fusion ECMs constructed in this study. The EREI2 fusion protein was 2 repeats of (APGVGV) 12 , RGD, (APGVGV) 12 and the IKVAV sequence.…”