کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5515952 | 1542299 | 2018 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae](/preview/png/5515952.png)
- Klebsiella pneumoniae FeoB (KpFeoB) was heterologously overexpressed in E. coli.
- Detergent-solubilized, purified KpFeoB is active towards GTP hydrolysis.
- The kcatGTP of KpFeoB suggests an active Fe2+ transport mechanism.
The acquisition of ferrous iron (Fe2+) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe2+ relative to Fe3+. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe2+ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (kcatGTP of â¼10â1 sâ1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe2+ import in an active manner.
Journal: Protein Expression and Purification - Volume 142, February 2018, Pages 1-7