Expression and purification of bioactive hemagglutinin protein of highly pathogenic avian influenza A (H5N1) in silkworm larvae

Dong, Jinhua; Harada, Mizuho; Yoshida, Sawako; Kato, Yuri; Murakawa, Akiko; Ogata, Makoto; Kato, Tatsuya; Usui, Taichi; Park, Enoch Y.

doi:10.1016/j.jviromet.2013.08.040

Cited by 8 publications

(2 citation statements)

References 25 publications

(25 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An important aspect of the system is the low cost and its relative simplicity252627282930313435. The cost of feeding the silkworms is very low, with a total cost for 20 larvae of about 20 US dollars.…”

Section: Discussionmentioning

confidence: 99%

Efficient and cost effective production of active-form human PKB using silkworm larvae

Maesaki

Satoh

Taoka

et al. 2014

Sci Rep

View full text Add to dashboard Cite

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

show abstract

Section: Discussionmentioning

confidence: 99%

Efficient and cost effective production of active-form human PKB using silkworm larvae

Maesaki

Satoh

Taoka

et al. 2014

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Moreover, lipid content in silkworm tissues, such as the fat body, is also abundant compared to that in other hosts used for protein expression [7,8]. 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].…”

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

Takamura

et al. 2020

Preprint

Self Cite

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.

show abstract

Purification of virus-like particles (VLPs) expressed in the silkworm Bombyx mori

Minkner

Park

2018

Biotechnol Lett

View full text Add to dashboard Cite

Virus-like particles (VLPs) are a promising and developing option for vaccination and gene therapy. They are also interesting as shuttles for drug targeting. Currently, several different gene expression systems are available, among which the silkworm expression system is known for its mass production capacity. However, cost-effective purification with high purity of the target protein is a particular bottleneck for this system. The present review evaluates the advances in the purification of VLPs, especially from silkworm larval hemolymph. Beginning with applicable pre-treatments for VLPs over to chromatography methods and quality control of the purified VLPs. Whereupon the main focus is on the different chromatography approaches for the purification, but the structure of the VLPs and their intended use for humans make also the quality control important. Within this, the stability of the VLPs which has to be considered for the purification is as well discussed.

show abstract

Expression and purification of bioactive hemagglutinin protein of highly pathogenic avian influenza A (H5N1) in silkworm larvae

Cited by 8 publications

References 25 publications

Efficient and cost effective production of active-form human PKB using silkworm larvae

Efficient and cost effective production of active-form human PKB using silkworm larvae

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

Purification of virus-like particles (VLPs) expressed in the silkworm Bombyx mori

Contact Info

Product

Resources

About