A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

Minkner, Robert; Xu, Jian; Zagst, Holger; Wätzig, Hermann; Kato, Tatsuya; Oltmann-Norden, Imke; Park, Enoch Y.

doi:10.1016/j.jchromb.2019.121964

“…Nevertheless, the efficiency of MNP3 for the purification of recombinant proteins from a complex matrix was proven, as a high protein recovery could be achieved. Especially as we already showed in other studies that high purity and high recovery by simultaneous high host cell reduction from the silkworm system is very challenging so far [ 12 , 13 ]. On the other hand, the purification is promising, but not satisfying enough.…”

Section: Resultsmentioning

confidence: 99%

“…The latter one is very intriguing, as the purification from this eucaryotic system is still troublesome and a big limitation for this system's usage. We reviewed the problems of the silkworm purification already elsewhere [ 7 ] and also showed that the purification is indeed not easy [ 12 , 13 ]. Therefore, we will not discuss this aspect exhaustively any further.…”

Section: Resultsmentioning

confidence: 99%

“…Usually, for purification from this system, traditional sucrose gradient density centrifugation and affinity purification are used, but generally, they have not been explored for optimization [ 9 – 11 ]. Approaches to improving purification with this system solely based on standard methods also present challenges [ 12 ]. Currently, 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.…”

Section: Introductionmentioning

confidence: 99%

“…The silkworm expression system is an example of a complex sample matrix. Therefore, purification from this system is still a bottleneck for the use of this expression system as it was already reviewed [ 7 ], and we could show that the purification is indeed not easy [ 12 , 13 ]. Therefore, we will not discuss this aspect exhaustively any further in this paper.…”

Section: Introductionmentioning

confidence: 99%

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

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Xu

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Takemura

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et al. 2020

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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.

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“…Usually, for puri cation from this system, traditional sucrose gradient density centrifugation and a nity puri cation are used, but generally, they have not been explored for optimization [9][10][11]. Approaches to improving puri cation with this system solely based on standard methods also present challenges [12]. Currently, no broadly practical puri cation approach is available for the puri cation of proteins from silkworms, except for some exceptional cases in which the host proteins are abundant and diverse.…”

Section: Introductionmentioning

confidence: 99%

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

Minkner

¹

,

Xu

²

,

Takamura

³

et al. 2020

Preprint

Self Cite

0

View full text Add to dashboard Cite

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.

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Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

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This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. “Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications,” pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

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A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

Cited by 7 publications

References 19 publications

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

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

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

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

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