Enhancing Solubility and Purification Efficiency of Vitronectin in Escherichia coli and Application in Human Pluripotent Stem Cell Culture

Abstract

Vitronectin (VTN) is a crucial protein substrate in human pluripotent stem cell (hPSC) culture, although challenges have been encountered in its bacterial expression and solubility. This study aimed to enhance the solubility and purification efficiency of VTN and its truncated forms, VTN(N) and VTN(NC), in the Escherichia coli expression system by fusion with tag proteins. Seven tags were evaluated, with MBP emerging as the most effective, significantly increasing the water solubility of VTN, VTN(N), and VTN(NC) from 5-60% to over 95%. The increased solubility of the fusion proteins, MBP-VTN(N) and MBP-VTN(NC), simplified the purification processes by removing solubilization and refolding steps of VTN. The purified fusion proteins also demonstrated superior stability during long-term storage. In a biological activity test, MBP-VTN(N) and MBP-VTN(NC) allowed for hPSC maintenance at the single cell level, a persistent challenge with VTN alone. Notably, the MBP-VTN(N) and MBP-VTN(NC) preserved cell morphology and pluripotency after several passages, outperforming VTN, which led to cell detachment after a few days. These findings suggest that the MBP-VTN(N) and MBP-VTN(NC) offer significant advantages for hPSC culture, paving the way for cost-effective and efficient stem cell research. Further studies are needed to optimize this approach and explore its full potential in stem cell biology.