Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

Bae, Sung Min; Kim, Hee Jung; Lee, Jun Beom; Choi, Jae Bang; Shin, Tae Young; Koo, Hyun‐Na; Choi, Jae Young; Lee, Kwang-Sik; Je, Yeon Ho; Jin, Byung Rae; Yoo, Sung Sik; Woo, Soo Dong

doi:10.1371/journal.pone.0060835

Cited by 13 publications

(13 citation statements)

References 37 publications

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“…The recombinant viruses were generated using these vectors and named rAcPCV2ORF2, rAc-19-110-PCV2ORF2, and rAc-32-85T-PCV2ORF2, respectively. Although fusion of polyhedrin 19-110 increased the yield of recombinant PCV2 ORF2, it was lower than that of enhanced green fluorescent protein (EGFP) (Bae et al, 2013). These results corresponded to previous results that showed that fusion the control of the AcMNPV polyhedrin promoter (Fig.…”

Section: Enhanced Production Of Recombinant Capsid Proteinsupporting

confidence: 90%

“…Recently, we demonstrated that the production of foreign proteins fused with various partial polyhedrin could enhance the production of foreign proteins significantly (Bae et al, 2013). Among various fused polyhedrin regions, fusions with amino acids 19-110 were able to localize the foreign proteins in the nucleus and enhance the expression level most highly.…”

Section: Construction Of Transfer Vectormentioning

confidence: 99%

“…However, cleavage of the recombinant protein when fused with polyhedrin 32-85 was not observed. This result suggested that the effect of partial polyhedrin fusion to the target protein may vary according to the target protein (Bae et al, 2013). The optimal multiplicity of infection (MOI) of virus and post infection time were determined by comparing various MOIs and harvesting times (Fig.…”

Section: Enhanced Production Of Recombinant Capsid Proteinmentioning

confidence: 99%

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Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Lee

Bae²,

Shin³

et al. 2015

International Journal of Industrial Entomology

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Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has typespecific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using antiHis monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

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Section: Enhanced Production Of Recombinant Capsid Proteinsupporting

confidence: 90%

Section: Construction Of Transfer Vectormentioning

confidence: 99%

Section: Enhanced Production Of Recombinant Capsid Proteinmentioning

confidence: 99%

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Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Lee

Bae²,

Shin³

et al. 2015

International Journal of Industrial Entomology

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“…At protein terminal level, hydrophilicity of histidine tag enhances the high solubility of expressed recombinant fusion proteins [8]. At protein level, polyhedrin is used as a carrier protein to facilitate antigen purification [9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Purification Propensity for Proteins from Bacillus halodurans

Yan¹,

Wu²

2016

Enz Eng

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The demand for proteins with special purposes increases significantly. These proteins are generally obtained through recombinant proteins, however their purification is costly and not easy. It is necessarily important to develop a method to estimate the chance of purification beforehand in order to have a prospective on proteins in question. Purification of a protein should be related to instinct properties of a protein including its 3D structure, and so far around 540 amino acid properties are found. Thus it is possible to test each amino acid property against the successful rate of protein purification to find out which property is more suitable to estimate the purification propensity. In this study, each of 535 properties was tested against 438 purified and 429 impossible purified proteins from Bacillus halodurans using logistic regression and neural network model. ROC analysis was applied to the resultant sensitivity and specificity. The results show that amino acid composition properties were generally less helpful to estimate the purification propensity whereas amino acid physicochemical properties, secondary structures and dynamic properties were more useful, and dynamic properties were more promising. Therefore several types of protein properties can serve to determine purification propensity of proteins, and have the potential to reduce the cost and to speed up the production in microbiological and biotechnical fields. Purification Propensity for Proteins from Bacillus halodurans Results and DiscussionIt is important for biotechnological industries to make a large quantity of highly stable and purified recombinant proteins, which provide economically affordable sources for clinical and industrial applications and research. Usually, purification is laborious and unexciting although various expression systems are employed successfully, such as codon optimization in expression. This is the reason why amino acid properties were analyzed to find out which amino acid property could provide a clue on the chance of successful purification.The upper panel of Figure 1 showed the accuracy, sensitivity and specificity resulting from logistic regression that was used to find out which of 535 amino acid properties was useful to estimate the purification propensity for 857 proteins from B. halodurans. In this figure, x-axis indicated each of 535 amino acid properties (Supplementary Material) while y-axis indicated the accuracy, sensitivity and specificity. At first glance, the specificity was the best followed by the accuracy and the sensitivity. Moreover, little difference appeared between 535 amino acid properties because the specificity, accuracy and sensitivity were colored similarly, but it was necessary to pick out the poorly performed amino acid properties, which were colored in blue in the upper panel of Figure 1. The amino acid properties related to electric charges were not good in estimating the chance of protein purification.The lower panel of Figure 1 displayed the receiver operating characterist...

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“…Polyhedrin has been used as a carrier protein to facilitate antigen purification [14-18]. This strategy for purification of antigen has been patented (see http://otl.sinica.edu.tw/en/index.php?t=9&group_id=19&article_id=477).…”

Section: Introductionmentioning

confidence: 99%

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

Ardisson-Araújo

Rocha

Hedil

et al. 2013

Virol J

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BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

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Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

Cited by 13 publications

References 37 publications

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Purification Propensity for Proteins from Bacillus halodurans

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

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