通过体外DNA混洗技术鉴定蛋白质域功能

Kathy Hiu Laam Po; Edward Wai Chi Chan; Sheng Chen

doi:10.21769/BioProtoc.2873

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Characterization of Protein Domain Function via in vitro DNA Shuffling

通过体外DNA混洗技术鉴定蛋白质域功能

KP Kathy Hiu Laam Po

EC Edward Wai Chi Chan

SC Sheng Chen email

发布: 2018年06月05日第8卷第11期 DOI: 10.21769/BioProtoc.2873 浏览次数: 5490

评审: Modesto Redrejo-RodriguezElizabeth LibbyChijioke Joshua

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Antimicrobial Agents and Chemotherapy, featuring study using the protocol.

Oct 2017

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

重组细菌效应子存在条件下ARP2/3磷酸化修饰的检测

Céline Michard [...] Patricia Doublet

2017年04月05日 8092 阅读

分枝杆菌中蛋白乙酰化状态的体外和体内检测

Krishna K. Singh [...] Deepak K. Saini

2019年07月05日 4809 阅读

嗜肺军团菌介导的泛素修饰和磷酸核糖泛素连接的体外谷氨酰化抑制

Alan G. Sulpizio [...] Yuxin Mao

2020年11月05日 2682 阅读

Abstract

We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the bla_CTX-M-14 and bla_CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.

Keywords: CTX-M-14 (CTX-M-14)

CTX-M-15 (CTX-M-15)

DNA shuffling (DNA混洗)

Hybrid enzyme (杂交酶)

Evolution (演化)

Background

DNA recombination is a natural process by which genetic materials are exchanged among bacteria to enhance survival fitness under environmental stresses. Several hybrid CTX-M-lactamases (CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132), presumably resulting from recombination between the bla_CTX-M-14 and bla_CTX-M-15 genes, the most common variants worldwide, have been reported in recent years (Nagano et al., 2009; Tian et al., 2014; He et al., 2015; Liu et al., 2015). Among these hybrid enzymes, CTX-M-64, which contained the N- and C-terminal portions of CTX-M-15 and the middle fragment of CTX-M-14, exhibited even higher catalytic activity than their parental prototypes (He et al., 2015).

DNA shuffling is a molecular approach designed to mimic and accelerate the evolution process through PCR-mediated random combinations of two target genes (Crameri et al., 1998). Our previous study demonstrated the use of DNA shuffling to investigate the molecular events driving the evolution of the CTX-M-type β-lactamases (Po et al., 2017). Mutants with or without cefotaximase activity were recovered. Important amino acid residues that played a role in conferring enzyme activity were identified by comparative analysis of the genotypes and phenotypes of the mutants. Such approach can be employed to characterize other functional proteins. Here we describe a detailed protocol of in vitro DNA shuffling.

Materials and Reagents

96-well cell culture plate (SPL Life Sciences, catalog number: 30096 )
Test tubes
Cotton swab (HUBY-340-CA-006)
Filter (Pall, catalog number: 4612 )
Petri dish (Corning, Gosselin^TM, catalog number: SB93-101 )
E. coli DH5α (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 12297016 )
E. coli BL21 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: C600003 )
pET15b
5x Green GoTaq^® Flexi Reaction Buffer (Promega, catalog number: M8911 )
2.5 mM dNTP mixture (Takara Bio, catalog number: 4030 )
Magnesium chloride (UniChem, catalog number: M04550-4G )
Primers (Synthesized by BGI)
rTaq (Takara Bio, catalog number: R001WZ )
PBS (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10010023 )
1x TAE (see Recipes)
Milli-Q water
QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
DNase I (Sigma-Aldrich, catalog number: DN25-1G )
GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: SM1331 )
Gel loading dye (New England Biolabs, catalog number: B7024S )
SacI-HF (New England Biolabs, catalog number: R3156S )
BamHI-HF (New England Biolabs, catalog number: R3136S )
CutSmart^® Buffer (New England Biolabs, catalog number: B7204S )
T4 DNA Ligase (New England Biolabs, catalog number: M0202S )
10x T4 DNA Ligase Buffer (New England Biolabs, catalog number: B0202S )
Tris (IBI Scientific, catalog number: IB70145 )
EDTA (Merck, catalog number: 1.08421.1000 )
Glacial acetic acid (DUKSAN, catalog number: 3839 )
Agarose, Molecular Biology Certified (IBI Scientific, catalog number: IB70045 )
10,000x Gold View I (West Gene, catalog number: WGO-1 )
LB Broth (Hopebio, catalog number: HB0128 )
LB Nutrient Agar (Hopebio, catalog number: HB0129 )
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-100G )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
Isopropyl-β-D-thiogalactopyranoside, IPTG (Santa Cruz Biotechnology, catalog number: sc-202185B )
Mueller-Hinton broth, MH broth (BD, BBL^TM, catalog number: 212322 )
Mueller-Hinton Agar, MH agar (Hopebio, catalog number: HB6232 )
Cefotaxime sodium salt (Sigma-Aldrich, catalog number: C7912-1G )
Sodium chloride (VWR, catalog number: VWRC27810.295 )
50 mM Magnesium chloride (see Recipes)
50x TAE buffer (see Recipes)
1% Agarose gel (see Recipes)
10x DNase I reaction mixture (see Recipes)
LB broth (see Recipes)
25 mg/ml Ampicillin (see Recipes)
0.5 M IPTG (see Recipes)
MH broth (see Recipes)
MH-IPTG (see Recipes)
MH agar (see Recipes)
Saline (see Recipes)

Equipment

Bio-Rad S1000^TM thermal cycler (Bio-Rad Laboratories, model: S1000^TM )
Centrifuge (Hettich Instruments, model: Mikro 185 )
Power pad for electrophoresis (Labnet International, model: ENDURO^TM 300V , catalog number: E0303)
Gel tank
UV transilluminator
Spectrophotometer (Hach, model: DR 2800^TM )
Conventional water bath (42 °C for transformation)
Conventional autoclave
Microwave
pH meter

Software

PyMOL software
BioEdit Sequence Alignment Editor

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Po, K. H. L., Chan, E. W. C. and Chen, S. (2018). Characterization of Protein Domain Function via in vitro DNA Shuffling. Bio-protocol 8(11): e2873. DOI: 10.21769/BioProtoc.2873.