Description
One of the major challenges to the economical preparation of biomedical and biotechnological recombinant proteins today resides in the availability of an efficient production and purification protocol. Some proteins may not lend themselves to effective purification or may require expensive and cumbersome multistep chromatographic protocols. On the other hand, use of synthetically produced protein based polymer fusion tags can allow for simplified, cost effective purification procedures and may also permit increased protein solubility, improved protein folding and/or increased protein production.
We have developed an elastin-like polymer (ELP) inspired by the mammalian elastin structure which consists of 220 repetitions of the pentapeptide VPAVG (V – valine; P – proline; A – alanine; G – glycine). This ELP presents a thermoresponsive behavior, which can be modulated by salt addition, as well as a high solubility which may be used as tools for efficient protein production and purification.
Objective: to demonstrate the potential of (VPAVG)220 as a tool for the simplified production and purification of recombinant proteins
Two high-value biomedical proteins: the antibacterial peptide CM4 (ABP-CM4) and the bone morphogenetic protein 2 (BMP-2) have been chosen as model proteins for this study. ABP-CM4 (3,8 KDa) is an alpha-helical peptide from Bombyx mori whose toxicity to the bacterial expression host and small size encumbers purification. BMP-2 (14,7 KDa) is a cytokine that triggers the development of mesenchymal stem cells into osteoblasts and is frequently insoluble when recombinantly produced.
The biopolymer tag VPAVG was fused to the C-terminus of both proteins with the inclusion of a formic acid cleavage site. Protein production was carried out in Escherichia coli BL21(DE3+) and purification was achieved by simple repetitive heating/cooling and centrifugation cycles. Formic acid treatment allowed tag removal and obtention of soluble proteins. Cleaved and non-cleaved BMP-2 were evaluated for cytotoxicity in C2C12 cell lines and cleaved ABP-CM4 was tested for its antimicrobial activity.
Future studies involve further investigation of the activity of these recombinantly produced proteins.
This study demonstrates the potential of using biopolymer fusion tags for the production and simplified purification of recombinant proteins.
; Costa, A. 
