肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征

李欣蓬; 杨林玉; 段昌园; 杨晓兰

doi:10.16016/j.1000-5404.202010006

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肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征

基础医学 | 更新时间：2026-01-09

- 肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征
- Immobilization and characterization of peptide deformylase on surface of ampholytic ion modified hydrophilic magnetic beads
- 第三军医大学学报 2021年43卷第6期页码：481-488
- 作者机构：
- 作者简介：
- 基金信息：
  
  国家自然科学基金面上项目(31570862);国家自然科学基金面上项目(81773625)
- DOI：10.16016/j.1000-5404.202010006
  中图分类号：
- 收稿：2020-10-05，
  
  纸质出版：2021-03-30
- 稿件说明：
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李欣蓬, 杨林玉, 段昌园, 等. 肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征[J]. 第三军医大学学报, 2021,43(6):481-488.

Xinpeng LI, Linyu YANG, Changyuan DUAN, et al. Immobilization and characterization of peptide deformylase on surface of ampholytic ion modified hydrophilic magnetic beads[J]. Journal of Third Military Medical University, 2021, 43(6): 481-488.
李欣蓬, 杨林玉, 段昌园, 等. 肽脱甲酰基酶在偶极离子修饰亲水磁珠表面的固定化及表征[J]. 第三军医大学学报, 2021,43(6):481-488. DOI： 10.16016/j.1000-5404.202010006.

Xinpeng LI, Linyu YANG, Changyuan DUAN, et al. Immobilization and characterization of peptide deformylase on surface of ampholytic ion modified hydrophilic magnetic beads[J]. Journal of Third Military Medical University, 2021, 43(6): 481-488. DOI： 10.16016/j.1000-5404.202010006.

摘要

Abstract

Objective

To compare the immobilization effect of 2 kinds of hydrophilic magnetic beads with Ni

-nitrilotriacetic acid (Ni

-NTA) group or carboxyl group and surface modified by ampholytic ions on

Mycobacterium tuberculosis

peptide deformylase (

PDF)

and to determine the target enzyme immobilization scheme appliable to screening

PDF ligand mixture library using magnetic separation combined with high performance liquid chromatography-mass spectrometry (HPLC-MS).

Methods

The plasmids pET-28a(+): def with N-terminal 6×His tagged

PDF was successfully constructed and transfected into

E.coli

BL21(DE3) for

PDF recombinant expression. The products were collected and purified using nickel-ion chelating affinity chromatography column

and then identified with the enzymatic properties. Then the obtained

PDF was coupled to magnetic beads with Ni

-NTA (

PDF-Ni

-NTA) or carboxyl group (

PDF-carboxy)

respectively. The immobilization capacity

enzyme activity

enzyme stability before and after immobilization

and the response to the inhibitor actinonin were compared between the 2 schemes.

Results

The immobilization capacity of

PDF-Ni

-NTA was 99.7±4.5 mg/g (

=3). When the ratio of amount of enzyme to magnetic beads was 1 ∶15

the retention activity of the immobilized

PDF was (107.2±13.9)% (

=3) of the free enzyme

and decreased to 43% after 3 h under shaking at 0 ℃ and pH value of 7.5. By contrast

the immobilization capacity of

PDF-carboxy was 36.9±2.6 mg/g (

=3)

and the retention activity of the immobilized

PDF reached to (102.6±15.4)% (

=3) when the ratio of amount of enzyme to magnetic beads was 1 ∶60

consistent with that on the Ni

-NTA magnetic beads

with negligible changes within 8 h under shaking at 0 ℃ and pH 7.5. The response of

PDF-carboxy to the inhibition of actinonin had no significant difference from that of free

PDF (

0.05).

Conclusion

PDF-carboxy magnetic beads present stable retention activity

and has similar inhibitory response as that of free

PDF

and thus is more suitable for screening of mixture library to discover high affinity ligands for

PDF.

关键词

Keywords

references

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