Ni-modified magnetic nanoparticles for affinity purification of His-tagged proteins from the complex matrix of the silkworm fat body

Abstract

Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.

Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

“…T22 (red box) is a cationic peptide targeting CXCR4 [53], L (brown box) is a peptidic linker (GGRSSRSS) that provides flexibility, GFP (green box) is an enhanced GFP and H6 is a hexahistidine tag (black box) [54,55]. H6 is useful for both chromatographic purification of the protein [54][55][56][57] and cation-mediated assembly [55,58]. N and C indicate the amino and carboxy-terminal ends, respectively.…”

Genetically engineered materials: Proteins and beyond

“…Proteins can also assemble into fiber-like 1D materials, some of which, with mass production and wide use, have generated long-lasting impacts on human society; about five millennia ago, China's civilization learned to raise Bombyx mori (silkworms) and to weave their delicate fibers into fine silk cloth, as well as substitute materials of paper for writing and recording. Empowered by modern genome engineering tools such as TALEN and CRISPR-Cas9, mankind now is able to genetically engineer the silkworms to produce silk materials with unprecedented properties, including those amenable to further covalent decoration by functional proteins both in vivo and in vitro [54][55][56]. Spider silk, another category of naturally occurring 1D protein material, is gaining traction with materials scientists in recent years for its exceptional mechanical and biochemical properties; dragline silk rivals steel in strength and is said to be nonimmunogenic and biocompatible for biomedical applications [57].…”

Aspects of high-performance and bio-acceptable magnetic nanoparticles for biomedical application

“…DspB is a glycoside hydrolase from Actinobacillus actinomycetemcomitans which helps in biofilm release [113] . Recently, Minkner et al investigated the use of Ni-modified MNPs for affinity purification of desired recombinant proteins expressed in the silk worm fat body [114] . Few examples of commercially available magnetic separators are Dynabeads™ magnetic cell separation and MagniSort technology from Thermo fisher and EasySep™ magnetic cell separation by stem cell technologies for the enrichment and isolation of cells from various species.…”