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Over-expression, rapid preparation and some properties of c-terminal BARc region in PICK1.

Xiao H, Shi Y, Yuan J, Huang Y, Wang J - Int J Mol Sci (2008)

Bottom Line: To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE.The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate.In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The First Affiliated Hospital, Shanxi Medical University, Taiyuan, P R China. xiaohh9999@yahoo.com.cn <xiaohh9999@yahoo.com.cn>

ABSTRACT
A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

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SDS-PAGE analysis for the gradient time test of MBP-BARc cleaved by human rhinovirus 3C protease. M: Protein marker; 0–20 h indicate the reaction time; Labeled bands correspond to MBP-BAR (74 kDa), MBP (42 kDa) and BARc (32 kDa) respectively.
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f3-ijms-10-00028: SDS-PAGE analysis for the gradient time test of MBP-BARc cleaved by human rhinovirus 3C protease. M: Protein marker; 0–20 h indicate the reaction time; Labeled bands correspond to MBP-BAR (74 kDa), MBP (42 kDa) and BARc (32 kDa) respectively.

Mentions: To remove the affinity tag from fusion protein MBP-BARc, an enzymatic cleavage procedure was conducted with human rhinovirus 3C protease under the condition as described in the Materials and Methods section. The result indicated from a gradient time test in Figure 3 that during human rhinovirus 3C protease cleavage, the BARc (about 32 kDa) was gradually released from the fusion product (about 74 kDa) until 20 h or so. The efficiency of removing MBP tag was about 95%, as analyzed by SDS-PAGE (Figure 3). The GDS analysis pointed out that the amount of BARc released from the fusion protein was in direct proportion with human rhinovirus 3C protease cleavage time. The result implies that the human rhinovirus 3C protease, a bench preparation, can efficiently cleave the corresponding tag fused to the target protein.


Over-expression, rapid preparation and some properties of c-terminal BARc region in PICK1.

Xiao H, Shi Y, Yuan J, Huang Y, Wang J - Int J Mol Sci (2008)

SDS-PAGE analysis for the gradient time test of MBP-BARc cleaved by human rhinovirus 3C protease. M: Protein marker; 0–20 h indicate the reaction time; Labeled bands correspond to MBP-BAR (74 kDa), MBP (42 kDa) and BARc (32 kDa) respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662470&req=5

f3-ijms-10-00028: SDS-PAGE analysis for the gradient time test of MBP-BARc cleaved by human rhinovirus 3C protease. M: Protein marker; 0–20 h indicate the reaction time; Labeled bands correspond to MBP-BAR (74 kDa), MBP (42 kDa) and BARc (32 kDa) respectively.
Mentions: To remove the affinity tag from fusion protein MBP-BARc, an enzymatic cleavage procedure was conducted with human rhinovirus 3C protease under the condition as described in the Materials and Methods section. The result indicated from a gradient time test in Figure 3 that during human rhinovirus 3C protease cleavage, the BARc (about 32 kDa) was gradually released from the fusion product (about 74 kDa) until 20 h or so. The efficiency of removing MBP tag was about 95%, as analyzed by SDS-PAGE (Figure 3). The GDS analysis pointed out that the amount of BARc released from the fusion protein was in direct proportion with human rhinovirus 3C protease cleavage time. The result implies that the human rhinovirus 3C protease, a bench preparation, can efficiently cleave the corresponding tag fused to the target protein.

Bottom Line: To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE.The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate.In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The First Affiliated Hospital, Shanxi Medical University, Taiyuan, P R China. xiaohh9999@yahoo.com.cn <xiaohh9999@yahoo.com.cn>

ABSTRACT
A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

Show MeSH
Related in: MedlinePlus