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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 of purified MBP-BARc, MBP and BARc. M: Protein marker; Lane 1: MBP-BARc; Lane 2: MBP-BARc cleaved by human rhinovirus 3C protease; Lane 3: MBP in the supernatant of 1.0 M (NH4)2SO4; Lane 4 and 5: re-soluble BARc in the precipitate at 1.0 M (NH4)2SO4.
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f4-ijms-10-00028: SDS-PAGE analysis of purified MBP-BARc, MBP and BARc. M: Protein marker; Lane 1: MBP-BARc; Lane 2: MBP-BARc cleaved by human rhinovirus 3C protease; Lane 3: MBP in the supernatant of 1.0 M (NH4)2SO4; Lane 4 and 5: re-soluble BARc in the precipitate at 1.0 M (NH4)2SO4.

Mentions: To separate the BARc from the enzymatic mixture of MBP-BARc, some methods such as ion exchange chromatography, gel filtration and even amylose re-affinity were attempted. Unfortunately, none was successful due to the aggregate of the sample, either BARc or MBP. In such case, we tried a classic method of ammonium sulfate precipitation and unexpectedly found that the BARc could be precipitated at 0.2—1.2 M (NH4)2SO4, while MBP was still soluble. The phenomenon may mean that the different aggregate state of MBP and BARc results in the different hydration shell round each protein. Finally, we used the concentration of 1.0 M (NH4)2SO4 to successfully separate the BARc from the enzymatic mixture. The BARc purity in the precipitate is about 95%, as analyzed by SDS-PAGE (Figure 4). Although the resoluble BARc may be contaminated with traces of MBP-BARc, it could be used as a sample for its functional tests.


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 of purified MBP-BARc, MBP and BARc. M: Protein marker; Lane 1: MBP-BARc; Lane 2: MBP-BARc cleaved by human rhinovirus 3C protease; Lane 3: MBP in the supernatant of 1.0 M (NH4)2SO4; Lane 4 and 5: re-soluble BARc in the precipitate at 1.0 M (NH4)2SO4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijms-10-00028: SDS-PAGE analysis of purified MBP-BARc, MBP and BARc. M: Protein marker; Lane 1: MBP-BARc; Lane 2: MBP-BARc cleaved by human rhinovirus 3C protease; Lane 3: MBP in the supernatant of 1.0 M (NH4)2SO4; Lane 4 and 5: re-soluble BARc in the precipitate at 1.0 M (NH4)2SO4.
Mentions: To separate the BARc from the enzymatic mixture of MBP-BARc, some methods such as ion exchange chromatography, gel filtration and even amylose re-affinity were attempted. Unfortunately, none was successful due to the aggregate of the sample, either BARc or MBP. In such case, we tried a classic method of ammonium sulfate precipitation and unexpectedly found that the BARc could be precipitated at 0.2—1.2 M (NH4)2SO4, while MBP was still soluble. The phenomenon may mean that the different aggregate state of MBP and BARc results in the different hydration shell round each protein. Finally, we used the concentration of 1.0 M (NH4)2SO4 to successfully separate the BARc from the enzymatic mixture. The BARc purity in the precipitate is about 95%, as analyzed by SDS-PAGE (Figure 4). Although the resoluble BARc may be contaminated with traces of MBP-BARc, it could be used as a sample for its functional tests.

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