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Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

Yu ZC, Tang BL, Zhao DL, Pang X, Qin QL, Zhou BC, Zhang XY, Chen XL, Zhang YZ - PLoS ONE (2015)

Bottom Line: With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h.In addition, another two cold-adapted enzymes were also successfully expressed by this system.Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.

ABSTRACT
Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

No MeSH data available.


Related in: MedlinePlus

Cellulase activity of Pseudoalteromonas sp. SM20429 harboring promoter reporter vectors p4xc or p4cc at different temperatures.The activity of the cellulase expressed by p4xc at 15°C for 72 h was taken as 100%.
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pone.0137384.g002: Cellulase activity of Pseudoalteromonas sp. SM20429 harboring promoter reporter vectors p4xc or p4cc at different temperatures.The activity of the cellulase expressed by p4xc at 15°C for 72 h was taken as 100%.

Mentions: Each of the reporter vectors (p4xc, p4cc or p4dc) was transferred into SM20429 and cultured at 5°C, 10°C and 15°C. The extracellular cellulase activity was measured after 24 h, 48 h and 72 h of cultivation. No cellulase activity was detected in the culture of SM20429 harboring p4dc, suggesting that the promoter of dnaK was most likely not suitable for protein expression in SM20429. At both 10°C and 15°C, SM20429 harboring p4xc showed a much higher cellulase activity than the transformants containing p4cc (Fig 2), indicating that the promoter of the xylanase gene is more stronger than the cspA promoter at a low temperature. Therefore, the promoter of the xylanase gene was chosen for protein expression in SM20429 at a low temperature.


Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

Yu ZC, Tang BL, Zhao DL, Pang X, Qin QL, Zhou BC, Zhang XY, Chen XL, Zhang YZ - PLoS ONE (2015)

Cellulase activity of Pseudoalteromonas sp. SM20429 harboring promoter reporter vectors p4xc or p4cc at different temperatures.The activity of the cellulase expressed by p4xc at 15°C for 72 h was taken as 100%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557933&req=5

pone.0137384.g002: Cellulase activity of Pseudoalteromonas sp. SM20429 harboring promoter reporter vectors p4xc or p4cc at different temperatures.The activity of the cellulase expressed by p4xc at 15°C for 72 h was taken as 100%.
Mentions: Each of the reporter vectors (p4xc, p4cc or p4dc) was transferred into SM20429 and cultured at 5°C, 10°C and 15°C. The extracellular cellulase activity was measured after 24 h, 48 h and 72 h of cultivation. No cellulase activity was detected in the culture of SM20429 harboring p4dc, suggesting that the promoter of dnaK was most likely not suitable for protein expression in SM20429. At both 10°C and 15°C, SM20429 harboring p4xc showed a much higher cellulase activity than the transformants containing p4cc (Fig 2), indicating that the promoter of the xylanase gene is more stronger than the cspA promoter at a low temperature. Therefore, the promoter of the xylanase gene was chosen for protein expression in SM20429 at a low temperature.

Bottom Line: With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h.In addition, another two cold-adapted enzymes were also successfully expressed by this system.Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.

ABSTRACT
Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

No MeSH data available.


Related in: MedlinePlus