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.
Highlights
For protein production, Escherichia coli or yeast systems are mainly utilized because they are already well established, and their advantages are known
The advantage of MNP3 is that stable magnetic separation could be achieved without the attenuation of the magnetism when a large amount of protein adhered to the particle surface
Magnetic separation of MNP3 from the solution was carried out using a magnet, and MNP3 was separated from the MNP3 solution at a concentration of 10 mg/ml in 10 s (Additional file 1: Fig. S3)
Summary
For protein production, Escherichia coli or yeast systems are mainly utilized because they are already well established, and their advantages are known. Low protein quality or the inability to generate posttranslational modifications are typical disadvantages [1]. Another interesting option for us is using the larvae of the domestic silkworm, Bombyx mori, as a recombinant protein expression system. These proteins make purification from the silkworm extremely challenging [1]. No broadly practical purification approach is available for the purification of proteins from silkworms, except for some exceptional cases in which the host proteins are abundant and diverse. We will not discuss this aspect exhaustively any further in this paper
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