Three Novel Escherichia coli Vectors for Convenient and Efficient

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Biotechnology and Biological Transformations

Three novel Escherichia coli vectors for convenient and efficient molecular biological manipulations Herui Gao, Xianghui Qi, Darren J. Hart, Song Gao, Hongling Wang, Shumin Xu, Yifeng Zhang, Xia Liu, Yifei Liu, and Yingfeng An J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01960 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Journal of Agricultural and Food Chemistry

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Three novel Escherichia coli vectors for convenient

2

and efficient molecular biological manipulations

3

Herui Gaoa§,Xianghui Qib§, Darren J. Hartc, Song Gaoa, Hongling

4

Wanga,Shumin Xua, Yifeng Zhanga, Xia Liua, Yifei Liua, Yingfeng Ana

§

§

*

5 6

a

7

Shenyang, China

8

b

9

China

10 11

c

College of Biosciences and Biotechnology, Shenyang Agricultural University,

School of Food and Biological Engineering, Jiangsu University, Zhenjiang,

Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes,

Grenoble 38044, France

12 13 14 15

§These authors contributed equally to this work

16 17

* Corresponding author: Yingfeng An

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Email: [email protected]

19

Address: College of Bioscience and Biotechnology, Shenyang Agricultural

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University. No.120 Dongling Road, Shenyang 110161, P. R. China.

21

Tel: +86-24-88487163. Fax: +86-24-88487163

1

ACS Paragon Plus Environment


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Abstract

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We have constructed novel plasmids pANY2, pANY3 and pANY6 for

24

flexible cloning with low false positives, efficient expression and convenient

25

purification of proteins. The pANY2 plasmid can be used for efficient

26

isopropyl-β-D-thiogalactoside (IPTG) induced protein expression, while the

27

pANY3 plasmid can be used for temperature-induced expression. The pANY6

28

plasmid contains a self-cleaving elastin-like protein (ELP) tag for purification of

29

recombinant protein by simple ELP-mediated precipitation steps and removal

30

of the ELP tag by self-cleavage. A urea-based denaturation and refolding

31

processes for renaturation of insoluble inclusion bodies can be conveniently

32

integrated into the ELP-mediated precipitation protocol, removing time

33

consuming dialysis steps. These novel vectors, together with the described

34

strategies of gene cloning, protein expression and purification, may have wide

35

applications in biosciences, agricultural and food technologies, etc.

36

Key words:

37 38

ELP; intein; Sticky-end ligation; TA cloning; Temperature-induced expression

39 40 41 42

2


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1. Introduction

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Molecular cloning is a fundamentally important step in many areas of

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biotechnology including genetic, agricultural and food technologies. In recent

46

years, with the rapid development of next-generation sequencing technologies,

47

the volume of genomic data is increasing rapidly. To begin to utilize such

48

important information in functional studies, more efficient high throughput

49

compatible tools for molecular biological manipulations are necessary.1

50

Sticky-end cloning is one of the most widely used strategies for molecular

51

cloning because the overhangs of plasmid vectors and DNA inserts can be

52

ligated with high efficiency.2 However, a high rate of false positives during

53

molecular cloning is a common problem which can significantly increase

54

workload of screening steps and reduce the quality of constructed DNA

55

libraries. T-vector-based TA cloning is another efficient strategy for cloning of

56

PCR products and is applicable to DNA fragments with unknown sequences

57

(e.g. complex DNA mixtures), a significant advantage over sticky-end cloning

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in some applications. The single 3′-T overhangs at both ends of the T-vectors

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can facilitate efficient cloning of DNA fragments with complementary 3′-A

60

overhangs.3 Until now, most commercially available T-vectors, such as

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pGEM-T Easy Vector (Promega) and pMDTM (TaKaRa), are designed for initial

62

cloning purposes only, meaning that further sub-cloning of the DNA inserts into

63

protein expression vectors is necessary for functional or structural studies. In

64

addition, many commercially available T-vectors are provided in linearized 3



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form and thus cannot be propagated further in E. coli, limiting their use and

66

increasing the cost of studies. Moreover, although phenotypic feature-based

67

screening (e.g. fluorescent proteins, β-galactosidase and toxic proteins) during

68

TA cloning may reduce workload of positive clone identification, problems may

69

arise due to inverted insertion of genes into T-vectors.2 Hemi-phosphorylation

70

of both T-vector and insert can be used to impart unidirectionality during TA

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cloning. To accomplish this, the DNA insert and vector backbone are produced

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by PCR using single phosphorylated PCR primers, or alternatively by digestion

73

with a first enzyme, dephosphorylation, then digestion with a second enzyme.4

74

However, these steps are time-consuming and reduce overall efficiencies.

75

A number of ligase-independent cloning methods have been developed, as

Gateway,5

LIC,6

SLIC,7

USER,8

CPEC,9

PIPE,10

OEC,11

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such

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FastCloning,12 SLiCE,13 and In-Fusion.14 Although efficient, they have not yet

78

supplanted ligase-dependent methods, in part due to the robustness and

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familiarity of the latter. Moreover, ligase-independent strategies are commonly

80

based on DNA homologous recombination necessitating a complicated design

81

of compatible regions between vectors and DNA inserts, or within DNA inserts.

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E. coli is commonly chosen for gene cloning and protein expression due to

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the wealth of knowledge about this organism and simple genetic manipulation.

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For example, E. coli has been chosen as a host for heterologous expression of

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various enzymes for industrial and agricultural applications,15 and expression

86

of proteins from various plants and plant pathogenic bacteria for functional 4


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analysis.16-20 There exist many expression vectors for expression of

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heterologous genes in E. coli, including pET,21 pTrc,22 pBAD.23 Each has its

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advantages and disadvantages, thus the choice of system should be

90

evaluated for each specific case.24 For applications such as structural biology

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that require high protein yields, pET vectors are popular, exploiting the

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powerful phage-derived T7 promoter. However, specific E. coli strains

93

expressing T7 polymerase are required and inducer concentrations (IPTG,

94

Arabinose) may need to be tuned to mitigate potential negative eﬀects

95

associated with the high viral polymerase activity.25 Temperature-inducible

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protein expression vectors (e.g., pBV220, pSW2, pKF396M-5) are convenient

97

because only shifting the culture temperature is required.26-28 However, most

98

established vectors are only available for sticky-end cloning with the limitations

99

described above. Also, a fixed induction temperature is recommended for

100

protein expression which may be problematic in some cases.29-31

101

Traditional affinity based purification methods for recombinant proteins

102

can be expensive on a large scale e.g. for industrial enzymes. Recently, a

103

non-chromatographic

104

elastin-like polypeptide (ELP) tags has been developed.32, 33 These precipitate

105

reversibly in response to mild increases in temperature at high salt

106

concentration allowing ELP-tagged proteins to be purified by selective

107

precipitation and centrifugation. In addition, self-cleaving inteins can be used in

108

conjunction with ELP tags to eliminate the need for expensive proteolytic tag

and

inexpensive

purification

5


method

based

on


109

removal.34-37 This ELP tag and intein-based purification strategy avoids

110

expensive affinity resins, columns and instruments. However, current ELP tag

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and intein-based vectors are formatted only for sticky end cloning, lacking the

112

advantages described above. In this study, we have designed and tested three

113

vectors (pANY2, pANY3 and pANY6) that provide a flexible cloning platform

114

compatible with multiple cloning approaches. These permit both IPTG- and

115

temperature-induced expression, and ELP-intein-based protein purification.

116

We have tested our new system using β-1,3-glucanase, an important enzyme

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with commercial applications. Furthermore, we demonstrate a potential

118

application in purifying material from insoluble inclusion bodies.

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2. Materials and methods

120

2.1. Construction of the vector pANY2

121

The plasmid pET3a was used as template for PCR amplifying the first

122

DNA fragment (Fragment I) using primers PHis-for1 (5′-AAAAC TGCAG

123

CACCA CCACC ACCAC CACTA AGGCT GCTAA CAAAG CCCGA AAGGA

124

AGCTG-3′; PstI underlined) and PstI-T-Rev (5′-GTAGT TTATC ACAGT

125

TAAAT TGCTA ACGCA GTCAG GGATA TCCGG ATATA GTTCC TCCTT

126

TC-3′). The plasmid pET9a was used as template for amplifying the second

127

DNA fragment (Fragment II) with primers P9A-for1 (5′-GAAAG GAGGA

128

ACTAT ATCCG GATAT CCCTG ACTGC GTTAG CAATT TAACT GTGAT

129

AAACT AC-3′) and P9a-rev1 (5′-CAAAG GCCAG CAAAA GGCCA GGAAC

130

CGTAA AAAGG CCGCG TTGCT GGCGT TT-3′). Then Fragment I and 6


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Fragment II were assembled by overlap extension PCR (OE-PCR) using

132

primers PHis-for1 and P9a-rev1 to give Fragment III. The plasmid pET3a was

133

used as template for amplifying Fragment IV using primers PT7-for2

134

(5′-GTTCC TGGCC TTTTG CTGGC CTTTG GTACC AGATC TCGAT CCCGC

135

GAAAT TAATA CGAC-3′) and PT7-rev2 (5′-AAAAC TGCAG CTGCA GCATA

136

TGTAT ATCTC CTTCT TAAAG TTAAA CAAAA TTATT TCTAG AGGG-3′).

137

Fragment IV and Fragment III were assembled by OE-PCR using primers

138

PHis-for1 and PT7-rev2 yielding Fragment V. This was digested with PstI,

139

gel-purified and performed self-cyclization using T4 DNA ligase. The ligation

140

product was transformed into E. coli JM109 to give plasmid pANY-orig. The

141

plasmid pHisKan5

142

primers CcdB-For2-middle (5′-GCCTG TCGAC CCTGG GTCTG GAGAC

143

CGGCT TACTA AAAGC CAGAT AACAG TATGC G-3′) and CcdB-Rev2-middle

144

(5′-CCAGG CCTGA CCATA GGTCG ACGAG AGACC GACTG GCTGT GTATA

145

AGGGA GCCTG AC-3′). Then the Fragment VI was used as template for

146

amplifying Fragment VII using primers CcdB-For2 (5′-CGCGC ATATG ACTAG

147

TAGGC CTGTC GACCC TGGGT CTGGA GACCG GC-3′; NdeI underlined)

148

and CcdB-Rev2 (5′-CATCTG CAGGA GCTCG GATCC AGGCC TGACC

149

ATAGG TCGAC GAGAG ACCGA CTGGC-3′; PstI underlined). Fragment VII

150

was digested with NdeI and PstI, ligated with similarly digested linearized

151

pANY-orig and the vector pANY2 recovered after transformation of E. coli

152

DB3.1. The plasmid pANY2 has been deposited to Addgene (No. 112196), and

38

was used as template for amplifying Fragment VI using

7



153

DNA sequence of pANY2 has been submitted to GenBank (No. MH385154).

154


155

Using plasmid pBV220 as template, PCR was performed with primers

156

TIE-KpnI-For (5′-CGGGG TACCG CGCCG ACCAG AACAC CTTGC

157

CGATC-3′) and TIE-NdeI-Rev (5′-CGCGC ATATG TTCCT CCTTA ATTTT

158

TAACC AATGC TTCG-3′). The product (Fragment VIII) was purified and

159

digested with the restriction endonucleases NdeI and KpnI, ligated with

160

similarly digested linearized pANY-orig and transformed into E. coli JM109

161

resulting in plasmid pANY-TIE. Fragment VII was digested with restriction

162

endonucleases NdeI and PstI, ligated with similarly digested linearized

163

pANY-TIE and the vector pANY3 recovered after transformation of E. coli

164

DB3.1. The plasmid pANY3 has been deposited to Addgene (No. 112197), and

165

DNA sequence of pANY2 has been submitted to GenBank (No. MH385155).

166


167

The plasmid pANY2 was used as template for amplifying the Fragment IX

168

using primers CcdB-His-For3 (5′-CGGGA TCCAC TAGTA GGCCT GTCGA

169

CGGTC AGTCC GGCTT ACTAA AAGCC AGATA ACAGT ATGCG-3′) and

170

CcdB-His-Rev3 (5′-CTGCA GGAGC TCCCA TGGAG GCCTG ACGGT

171

CAGTC GACTG GCTGT GTATA AGGGA GCCTG ACATT TATAT TC-3′).

172

Fragment X was amplified from pANY2 with primers pANY2-For1 (5′-GTCGA

173

CTGAC CGTCA GGCCT CCATG GGAGC TCCTG CAGTT GGCTG CTGCC

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ACCGC TGAGC AATAA CTAG-3′) and pANY2-Rev1 (5′-CGCGC ATATG 8


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TATAT CTCCT TCTTA AAGTT AAACA AAATT ATTTC TAGAG GGAAA

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CCG-3′). Fragments IX and X were assembled by OE-PCR using primers

177

CcdB-His-For3 and pANY2-Rev1 to give Fragment XI. An ELP sequence

178

encoding seven repeat units of nine successive VPGXG (i.e., Fragment XII)

179

was synthesized, in which the nine X residues were replaced by lysine, seven

180

valines and phenylalanine. Fragment XII was used as template for amplifying

181

the Fragment XIII using primers ELP-For1 (5′-CGCGC ATATG GTCCC

182

GGGGA AAGGC GTGCC TGGTG TCGGG GTTCC AGGTG-3′) and

183

ELP-Rev1 (5′-GTTGT TGTTA TTGTT ATTGT TGTTG TTGTT CGAGC TCACA

184

CCGAA TCCGG GGACC CCGAC ACCCG G-3′). Plasmid pET/EI-GFP36 was

185

used as template for amplifying XIV using primers Intein-For1 (5′-AGCTC

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GAACA ACAAC AACAA TAACA ATAAC AACAA CGAAT TCGCC CTCGC

187

AGAGG GCACT CGGAT C-3′) and Intein-Rev1 (5′-CGGGA TCCGT TGTGT

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ACAAC AACCC CTTCG GCGAC GAGGG TGTGC AGTTC CTCGA CCTCG

189

AG-3′). Fragments XIII and XIV were assembled by OE-PCR using ELP-For1

190

and Intein-Rev1 to give Fragment XV. The PCR product of Fragment XV was

191

digested with restriction endonucleases NdeI and BamHI, ligated with similarly

192

digested Fragment XI and the vector pANY6 recovered after transformation of

193

E. coli DB3.1. The plasmid pANY6 has been deposited to Addgene (No.

194

112198), and DNA sequence of pANY2 has been submitted to GenBank (No.

195

MH385156).

196

2.4. The vector pANY2 used for BsaI-based sticky-end cloning 9



197

The plasmid pET9d-pfLamA encoding endo-β-1,3-glucanase pfLamA39

198

was used as template to amplify the pfLamA gene using primers

199

pfLamA-For-BsaI (5′-GATCG GTCTC GGTCT ATGGT CCCTG AAGTG

200

ATAGA AATAG ATGGA AAACA G-3′) and pfLamA-Rev-BsaI (5′-GTACG

201

GTCTC TGACG ACCAC TAACG AATGA GTAAA CCCTT ACATA ATCC-3′).

202

Both pfLamA gene and pANY2 were digested with BsaI, ligated by T4 DNA

203

ligase, and transformed into E. coli JM109 to give recombinant plasmid

204

pANY2-sticky-pfLamA. Plasmids were confirmed by DNA sequencing

205

(GENEWIZ Inc., Suzhou, China).

206

2.5. The vector pANY2 used for unidirectional TA cloning

207

The plasmid pANY2 was used for unidirectional TA cloning. The plasmid

208

pET9d-pfLamA was used as template to amplify β-1,3-glucanase pfLamA gene

209

by PCR using Taq DNA polymerase, and the primers used for PCR were

210

pfLamA-For-AhdI (5′-GGCAT GGTCC CTGAA GTGAT AGAAA TAGAT

211

GGAAA ACAG-3′) and pfLamA-Rev-AhdI (5′-AGGAC CACTA ACGAA TGAGT

212

AAACC CTTAC ATAAT CC-3′). The plasmid pANY2 was digested with

213

restriction enzyme AhdI to give T-vector named pANY2-T. Then 30 ng pfLamA

214

and pANY2-T were mixed with 175 U T4 DNA ligase, 1×ligation buffer, 5 U

215

AvrII and 5 U NcoI in a final reaction volume of 20 µL. After incubation at 37 ℃

216

for 2 h, the mixture was used to transform E. coli JM109 to give recombinant

217

plasmid pANY2-TA-pfLamA. Plasmids were confirmed by DNA sequencing.

218

2.6. Construction of pANY3-pfLamA and pANY3-Hj-Xyn for 10


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temperature-induced expression

220

The endo-β-1,3-glucanase gene pfLamA and beta-xylanase gene Hj-Xyn

221

were inserted into pANY3. The plasmid pANY3 was digested with restriction

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enzyme AhdI resulting in a T-vector named pANY3-T. Using plasmid

223

pET9d-pfLamA as template, the pfLamA gene was amplified by PCR using Taq

224

DNA polymerase and primers pfLamA-For-AhdI (5′-GGCAT GGTCC CTGAA

225

GTGAT AGAAA TAGAT GGAAA ACAG-3′) and pfLamA-Rev-AhdI (5′-AGGAC

226

CACTA ACGAA TGAGT AAACC CTTAC ATAAT CC-3′). After purification, the

227

pfLamA gene was ligated with pANY3-T and the reaction used to transform E.

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coli JM109 to give plasmid pANY3-pfLamA. The Hypocrea jecorina

229

beta-xylanase gene Hj-Xyn was inserted into pANY3 through sticky-end

230

cloning. Using a synthetic Hj-Xyn gene as template, PCR was performed using

231

primers Hj-Xyn-NdeI-For (5′-GGAGG TAAAA CATAT GACGA TCCAA CCAGG

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CACGG GCTAC AACAA CGG-3′) and Hj-Xyn-PstI-Rev (5′-AAAAC TGCAG

233

GCTAA CGGTG ATGCT TGCAG AACCG CTGCT G-3′). After purification, the

234

Hj-Xyn gene was digested with NdeI and PstI, ligated with similarly digested

235

linearized pANY3 vector and plasmid pANY3-Hj-Xyn recovered following

236

transformation of E. coli JM109.

237

2.7. Screening positive clones by a protocol of bacterial lysate

238

electrophoresis

239

After transformation of E. coli JM109 with the ligation product for the

240

construction of the pANY3-pfLamA described above, ten transformants were 11



241

randomly selected and analyzed using bacterial lysate electrophoresis for fast

242

screening of positive clones. Each fresh transformant was inoculated in

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round-bottom Eppendorf tube containing 1 mL of LB medium supplemented

244

with 100 mg/L of kanamycin, and incubated in flask at 37 °C with shaking (220

245

rpm) for 5 h. Cell pellet was collected by centrifugation and resuspended in

246

100 µL resuspension buffer containing 10 mmol/L glucose, 0.5 mmol/L

247

Tris-HCl (pH 8.0) and 2 mmol/L EDTA. Then 40 µL lysis buffer containing 1%

248

SDS and 0.2 mol/L NaOH was added into the tube and the tube was gently

249

turned upside down for three times. Finally 100 µL phenol/chloroform/isoamyl

250

alcohol (25:24:1) was added to the tube followed by centrifugation at 12000×g

251

for 10 min. The supernatant was carefully decanted and subjected to agarose

252

gel electrophoresis. Plasmid DNAs were extracted from these randomly

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selected transformants and their inserts confirmed by DNA sequencing

254

following purification by miniprep.

255

2.8. Construction of pANY6-pfLamA for ELP-intein-based

256

purification

257

Using plasmid pET9d-pfLamA as template, the pfLamA gene was

258

amplified by PCR using Pfu DNA polymerase and primers pfLamA-for-NdeI

259

(5′-ACGCG CATAT GGTCC CTGAA GTGAT AGAAA TAGAT GGAAA ACAGT

260

GG-3′) and pfLamA-PstI (5′-CTAAC TGCAG TTAAC CACTA ACGAA TGAGT

261

AAACC CTTAC ATAAT CCACC-3′). The product was digested with NdeI and

262

PstI and ligated into similarly digested pANY6 vector, yielding pANY6-pfLamA. 12


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2.9. The vector pANY2 used for protein expression and

264

purification

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E. coli BL21 (DE3) was transformed with pANY2-TA-pfLamA and

266

pANY2-sticky-pfLamA. As a control, the plasmids pETM11-pfLamA (pfLamA

267

gene cloned into pETM11) and pETM11 were also used to transform E. coli

268

BL21 (DE3). For protein expression and purification, the transformants were

269

cultured in TB media, and expression of pfLamA was induced by 0.2 mmol/L

270

IPTG. The cells were pelleted by centrifugation, resuspended in 50 mmol/L

271

Tris-HCl buffer (pH 8.0), and then disrupted by sonication. The expressed

272

proteins were purified using HIS GraviTrap Ni-NTA agarose chromatography

273

(Chaoyan Biotechnology Co., Shanghai) and checked by SDS-PAGE. Ten

274

microliters of purified proteins were dispensed onto the surface of a LB plate

275

containing 1% curdlan (β-1,3-glucan); the endo-β-1,3-glucanase activity of

276

pfLamA was detected following staining with Congo red. The glycosyl

277

hydrolysate of curdlan was produced in 150 mmol/L citrate buffer (pH 5.0)

278

containing 1% curdlan and purified protein and at 80 ℃ for 30 min. The

279

reducing sugar products were detected by 3,5-Dinitro-2-hydroxybenzoic acid

280

(DNS) assay.40

281

2.10. The pANY3 vector used for temperature-induced protein

282

expression and purification

283

E. coli BL21 (DE3) was transformed with plasmids pANY3-pfLamA,

284

pANY3-Hj-Xyn and pETM11. For protein expression and purification, the 13



285

transformants were cultured in TB media supplemented with 100 mg/L of

286

kanamycin. Pre-cultures were grown at 30 ℃ and protein expression of each

287

protein was induced at various temperatures ranging from 32 to 45 ℃. The

288

expressed proteins were purified using Ni-NTA agarose chromatography

289

(Chaoyan Biotechnology Co., Shanghai). The protein samples were checked

290

by SDS-PAGE. The efficiency of pfLamA was detected by degradation degree

291

of curdlan in 150 mmol/L citrate buffer (pH 5.0) containing 1% curdlan and 19

292

mg/L purified pfLamA at 80 ℃ for 2 h. After centrifugation, the pellets of solid

293

curdlan samples with and without enzyme treatment were weighed. The

294

activity of Hj-Xyn was detected by degradation of xylan in 100 mmol/L sodium

295

citrate buffer (pH 5.0) containing 1% xylan and Hj-Xyn at 50 ℃ for 10 min.

296

The products were detected by DNS assay.

297

2.11. The pANY6 vector used for ELP-intein-based protein

298

purification

299

E. coli BL21 (DE3) was transformed with pANY6-pfLamA and pETM11.

300

Transformants were cultured in TB media supplemented with 100 mg/L of

301

kanamycin, and expression of pfLamA was induced by 0.2 mmol/L IPTG. Then

302

1 g cells of each strain was pelleted by centrifugation, and resuspended in 10

303

mL Lysis buffer (10 mmol/L Tris-HCl, 2 mmol/L EDTA, 0.1 g/L lysozyme, 0 or 8

304

mol/L Urea, pH 8.5), and then disrupted by sonication. The sonicated

305

suspensions were centrifuged at 12,000 × g for 10 min, and 200 µL

306

supernatants were mixed with 0.2-2 mol/L (NH4)2SO4 and incubated at 25-37 ℃ 14


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for 10 min. Then the samples were centrifuged at 25 ℃ for 10 min at 12,000

308

×g and the pellets resuspended in 150 µL cleaving buffer (PBS buffer

309

supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.0-8.5), and

310

incubated at 37 ℃ for 12 h. The samples were then mixed with 150 µL 0.2-2

311

mol/L (NH4)2SO4 or 0.2-2 mol/L NaCl, and incubated at 37 ℃ for 30 min, and

312

the purified proteins were obtained by centrifugation at 25 ℃ for 10 min at

313

12,000×g. The protein samples were checked by SDS-PAGE. The activity of

314

pfLamA was assayed in 150 mmol/L citrate buffer (pH 5.0) containing 1%

315

curdlan and purified protein at 80 ℃ for 10 min. The reducing sugar products

316

were detected by DNS assay. Protein concentrations were determined by

317

Bradford protein assay (Bio-Rad, Hercules, CA, USA). In this study, when

318

comparisons of activities (or values) under different conditions were performed,

319

the maximum activities (or values) were taken as 100% and the ratio between

320

the activities (or values) under different conditions and the corresponding

321

maximum activities (or values) were shown as relative activities (or values).

322

3. Results and Discussion

323

In this study, we have constructed novel E. coli vectors pANY2, pANY3

324

and pANY6 for efficient molecular cloning, protein expression and purification

325

using varied strategies. The ccdB cassette is used as a counter-selectable

326

marker and is flanked by two multiple cloning sites (MCS1 and MCS2). MCS1

327

and MCS2 of pANY2 contain commonly used restriction sites (e.g., NdeI, SpeI,

328

SacI, BamHI, StuI) (Fig. 1-a). Moreover, there are two BsaI sites permitting 15



329

sticky-end cloning (Fig. 1-b) and two AhdI sites for TA-cloning.

330

3.1. BsaI-based sticky-end cloning

331

BsaI-based sticky-end cloning avoids false positives via a mechanism

332

illustrated in Fig. 2-a. The pANY2 vector and PCR product of DNA insert are

333

digested with BsaI and ligated with T4 DNA ligase. BsaI recognizes a

334

non-palindromic sequence 5′-GGTCTCN↓NNNNN-3′ thus the overhangs can

335

be specifically designed to avoid intramolecular self-ligation or intermolecular

336

self-ligation of linearized vector (Fig. 2-b). Moreover, contamination by the

337

parental pANY2 plasmid can be avoided via the selectable ccdB cassette. All

338

plasmids from ten randomly selected transformants were positive by DNA

339

sequencing.

340

3.2. Unidirectional TA cloning

341

AhdI recognizes 5′-GACNNN↓NNGTC-3′ and cuts to generate a single

342

base overhang at the 3′ end; thus it can be used to introduce single T

343

overhangs at both termini of the linearized vector. In this way, a T-vector was

344

prepared by AhdI digestion of pANY2 and efficiency demonstrated by

345

unidirectional cloning of the pfLamA gene (Fig. 3). Both pANY2 and insert

346

contain part of the recognition sequences of AvrII and NcoI with full sites being

347

generated by ligation of the insert in the undesired orientation. Consequently,

348

inclusion of AvrII and NcoI in the ligase reaction results only in unidirectional

349

TA cloning. Commonly, positive clones from TA cloning are identified by

350

blue-white screening on X-gal plates; however, the lacZα gene may not be 16


Page 16 of 43

Page 17 of 43


351

disrupted by small DNA inserts resulting in false α-complementation.41 Use of

352

the counter-selectable ccdB cassette avoids this problem. Here, DNA

353

sequencing confirmed correct ligation of the pfLamA insert in all ten randomly

354

selected transformants.

355

3.3. The pANY2 vector used for IPTG-induced protein

356

expression and purification

357

Most commercially available T-vectors are designed for primary cloning of

358

DNA inserts, requiring further subcloning into protein expression vectors if

359

necessary. By contrast, the T-vector pANY2 can be used for both efficient

360

insert cloning and expression for subsequent purification, as exemplified here

361

with the endo-β-1,3-glucanase gene pfLamA. For comparison, both pANY2

362

and pET11 were used as vectors for expression and purification of pfLamA.

363

Respectively, 2.7 µg, 7.9 µg and 3.3 µg pfLamA protein samples were purified

364

from 1 mL cultures of E. coli strains harboring plasmids pET11-pfLamA,

365

pANY2-TA-pfLamA and pANY2-sticky-pfLamA. The expression level of

366

pfLamA from pANY2-TA-pfLamA was higher than that from pET11-pfLamA;

367

with pANY2-sticky-pfLamA it was lower with some degradation visible by

368

SDS-PAGE. Thus, in some cases, expression level and stability of a protein

369

might vary due to changing of fusion proteins and even cloning sites (Fig. 4-a)

370

since these modify the amino acids at the termini of the protein sequence.

371

Small variations at the termini of a protein affect the level of expression and

372

stability of the protein in an unpredictable manner.42 The activity of expressed 17



Page 18 of 43

373

pfLamA was tested using the Congo red agar method with clear zones forming

374

in curdlan agar (Fig. 4-b). The activity of purified pfLamA was further verified by

375

DNS assay (Fig. 4-c). The specific activity of the purified pfLamA was about

376

6.3 units/mg, where one unit is defined as the amount of enzyme that release 1

377

µmole of glucose per minute at pH 5.0 and 80 ℃.

378

3.4. Construction of pANY3 and pANY6, and identification of

379

positive clones via bacterial lysate electrophoresis

380

In contrast to the T7 promoter-based expression system of pANY2,

381

pANY3

contains

the

promoter

system

382

temperature-induced expression (Fig. 5-a). The performance of pANY3 for

383

cloning was exemplified using the endo-β-1,3-glucanase gene pfLamA. A

384

faster and more convenient protocol of bacterial lysate electrophoresis was

385

used to screen positive clones during pANY3-pfLamA construction yielding

386

clear bands consistent with the size of the extracted plasmid (Fig. 5-b). It is

387

common knowledge that even after careful extraction, a plasmid should be a

388

mixture of supercoiled, nicked circular and linear DNAs, and supercoiled DNA

389

generally travels through agarose more quickly than does the nicked or linear

390

forms during agrose gel electrophoresis. However, in this study, when the main

391

form of a plasmid (i.e., supercoiled form) was used for comparison after

392

bacterial lysate electrophoresis, it was very easy to distinguish between empty

393

plasmids and recombinant plasmids. This protocol is similar to the alkaline

394

lysis method, but omits ethanol precipitation and wash steps. This avoids 18


of

pL/pR/Cits857

for

Page 19 of 43


395

repeated steps of cleaning DNA that are time-consuming especially when

396

high-throughput preparation of DNA samples is required. Most of the tested

397

colonies (9/10) were positive for inserts with plasmids migrating at a different

398

molecular weight to the parent vector (Fig. 5-b), further confirmed by DNA

399

sequencing.

400

The plasmid pANY6 contains an ELP tag for protein purification by

401

reversible precipitation, and a self-cleaving intein for removal of ELP tag by

402

self-cleavage (Fig. 5-c). The protein purification strategy is illustrated in Fig.

403

5-d. The ELP-intein-TP fusion protein is extracted from cells and reversibly

404

precipitated at high salt concentration. The TP is separated from the

405

ELP-intein moiety by self-cleavage at low pH.

406

3.5. The performance of pANY3 in temperature-induced

407

protein expression

408

E. coli BL21 (DE3) was transformed with pANY3-pfLamA and

409

pANY3-Hj-Xyn for temperature-induced expression. The crude and purified

410

pfLamA fractions from different strains were analyzed by SDS-PAGE (Fig. 6-a).

411

Observed yields were 1.9 µg and 1.7 µg proteins purified from 1 mL cultures

412

with pANY3-pfLamA and pANY3-Hj-Xyn respectively, indicating that both

413

pfLamA and Hj-Xyn are well expressed with this system. The activity of

414

expressed pfLamA was detected through degradation of curdlan. Under the

415

optimized conditions, 21% of curdlan remained insoluble after treatment with

416

the expressed pfLamA, indicating efficient catalysis (Fig. 6-b). The activity of 19



Page 20 of 43

417

Hj-Xyn was verified by DNS assay (Fig. 6-c). The specific activity of the

418

purified Hj-Xyn was about 7.5 units/mg, where one unit is defined as the

419

amount of enzyme that release 1 µmole of xylose per minute at pH 6.0 and

420

39 ℃. Commonly 42 ℃ is recommended as a fixed induction temperature for

421

temperature-inducible expression vectors.29-31 In this study, the expression of

422

these enzymes at different temperatures was performed to determine the

423

optimum induction temperatures (Fig. 6-d). The results indicated that 42 ℃

424

and 39 ℃ were optimal for pfLamA and Hj-Xyn, respectively. For pfLamA, the

425

differences in protein expression levels from 37 ℃ to 45 ℃ varied less than

426

20%. However, for Hj-Xyn, protein expression decreased sharply when

427

induction temperatures were higher or lower than 39 ℃ . These results

428

suggest the induction temperature is an important variable that may need to be

429

optimized for each target protein when using this system.

430

3.6. The performance of pANY6 used for ELP-intein-based

431

purification of partially soluble protein pfLamA

432

The effects of pH ranging from 6.0 to 8.5 on intein cleavage efficiency of

433

ELP-intein-pfLamA

434

decreasing pH, and was complete at pH 6.0 (Fig. 7-a). The optimal

435

concentrations of (NH4)2SO4 and NaCl were also investigated. Precipitation

436

increased

437

Concentrations of 0.7 mol/L (NH4)2SO4 and 3.5 mol/L NaCl proved optimal in

438

terms of the amount of precipitated ELP-intein-pfLamA versus the precipitation

with

were

investigated.

decreasing

The

(NH4)2SO4

efficiency

and

20


NaCl

increased

with

concentrations.

Page 21 of 43


439

of contaminant proteins (Fig. 7-b, c). The phase transition temperatures of

440

ELP-intein-pfLamA was 32.4 ℃ (Fig. 7-d), with nearly all the target protein

441

precipitating at 45 ℃. Under optimized conditions, about 2.2 µg pfLamA

442

protein could be purified from a 1 mL culture of E. coli [pANY6-pfLamA].

443

Proteins expressed in E. coli often form insoluble inclusion bodies that can

444

sometimes be addressed by denaturation of the bodies with urea, then

445

refolding by removal of urea by dialysis. This refolding process is performed

446

prior to downstream chromatographic purification steps, and is both

447

time-consuming and often unsuccessful. We modified the ELP-intein-based

448

purification procedure to address insoluble inclusion bodies by combining

449

denaturation-refolding and protein purification in a single step. The urea is

450

added directly to the cell lysis buffer to denature and solubilize the inclusion

451

bodies; the target protein is then precipitated in high salt concentrations and

452

via a pH shift. The practicality and efficiency of this protocol was demonstrated

453

by purification of pfLamA from ELP-intein-pfLamA which is expressed in a

454

mainly insoluble form in E. coli. Addition of urea resulted in a 3-fold increase in

455

the final concentration, and 2-fold increase of the total activity, of purified

456

pfLamA (Fig. 7-e). This protocol should be applicable to many insoluble

457

proteins expressed in E. coli.

458

In summary, we have developed three novel vectors (pANY2, pANY3 and

459

pANY6), which are simple to use and result in high cloning efficiencies using

460

different cloning strategies. The vector pANY2 can be used for IPTG-induced 21



461

protein expression, while pANY3 permits temperature-induced expression.

462

The plasmid pANY6 can be used for ELP-intein-based purification of soluble

463

proteins, and has further applications when proteins are expressed insolubly,

464

combining denaturation-refolding and protein purification into a single

465

convenient step. These vectors and associated protocols should find wide

466

applications in many fields, including biosciences, agriculture and food

467

technologies.

468

Acknowledgments

469

The authors would like to thank Sergi Castellano and Promdonkoy

470

Patcharee for helpful discussions and review of this manuscript.

471

Funding Sources

472

This work was supported by National Natural Science Foundations of

473

China (grant numbers 31100045, 31270114, 31571806), Program for Liaoning

474

Excellent Talents in University (grant number LR2014018), and Liaoning

475

BaiQianWan Talents Program (grant number 2015-40).

476 477 478 479 480 481 482

References (1) Celie, P.H.; Parret, A.H.; Perrakis, A. Recombinant cloning strategies for protein expression. Curr. Opin. Struct. Biol. 2016,38,145-154. (2) Yao, S.; Hart, D.J.; An, Y. Recent advances in universal TA cloning methods for use in function studies. Protein Eng. Des. Sel. 2016,29,551-556. (3) Aranishi, F.; Okimoto, T. Engineered XcmI cassette-containing vector 22


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for PCR-based phylogenetic analyses. J. Genet. 2004,83,33-34. (4) Zhou, M.Y.; Gomez-Sanchez, C.E. Universal TA cloning. Curr. Issues

484 485

Mol. Biol. 2000,2,1-7. (5) Hartley, J.L.; Temple, G.F.;Brasch, M.A. DNA cloning using in vitro

486 487

site-specific recombination. Genome Res. 2000,10,1788-1795.

488

(6) Bonsor, D.; Butz, S.F.; Solomons, J.; Grant, S.;Fairlamb, I.J.; Fogg,

489

M.J.; Grogan, G. Ligation independent cloning (LIC) as a rapid route to families

490

of recombinant biocatalysts from sequenced prokaryotic genomes. Org.

491

Biomol. Chem. 2006,4,1252-1260. (7) Li, M.Z.; Elledge, S.J. Harnessing homologous recombination in vitro to

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generate recombinant DNA via SLIC. Nat. Methods 2007,4,251-256.

494

(8) Geu-Flores, F.; Nour-Eldin, H.H.; Nielsen, M.T.; Halkier, B.A. USER

495

fusion: a rapid and efficient method for simultaneous fusion and cloning of

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multiple PCR products. Nucleic Acids Res. 2007,35,e55. (9) Quan, J.; Tian, J. Circular polymerase extension cloning of complex

497 498

gene libraries and pathways. PLoS One 2009,4,e6441.

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(10) Klock, H.E.; Lesley, S.A. The Polymerase Incomplete Primer

500

Extension (PIPE) method applied to high-throughput cloning and site-directed

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mutagenesis. Methods Mol. Biol. 2009,498,91-103. (11) Bryksin, A.V.; Matsumura, I. Overlap extension PCR cloning: a simple

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2010,48,463-465.

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(12) Li, C.; Wen, A.; Shen, B.; Lu, J.; Huang, Y.; Chang, Y. FastCloning: a

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highly simplified, purification-free, sequence- and ligation-independent PCR

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cloning method. BMC Biotechnol. 2011,11,92.

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(13) Zhang, Y.; Werling, U.; Edelmann, W. SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res.2012,40,e55. (14) Sleight, S.C.; Sauro, H.M.BioBrick™ assembly using the In-Fusion PCR Cloning Kit. Methods Mol. Biol. 2013,1073,19-30.

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(15) Xing, M.N.; Zhang, X.Z.; Huang, H. Application of metagenomic

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techniques in mining enzymes from microbial communities for biofuel

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(16) Bryant, D.; Cummins, I.; Dixon, D.P.; Edwards, R. Cloning and

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characterization of a theta class glutathione transferase from the potato

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pathogen Phytophthora infestans. Phytochemistry. 2006,67,1427-1434.

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(17) Che, Y.Z.; Li, Y.R.; Zou, H.S.; Zou, L.F.; Zhang, B.; Chen, G.Y. A novel

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antimicrobial protein for plant protection consisting of a Xanthomonas oryzae

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harpin and active domains of cecropin A and melittin. Microb. Biotechnol.

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2011,4,777-793.

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(18) Du, X.; Zhang, X. Molecular cloning and functional characterization of

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two novel high molecular weight glutenin subunit genes in Aegilops markgrafii.

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J. Genet. 2017,96,563-570.

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(19) Liu, C.; Zheng, K.; Xu, Y.; Stephen, L.T.; Wang, J.; Zhao, H.; Yue, T.;

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Nian, R.; Zhang, H.; Xian, M.; Liu, H. Expression and characterization of

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soybean seed coat peroxidase in Escherichia coli BL21(DE3). Prep. Biochem.

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Kurosaki, F.; Lee, J.B. Characterization of ent-kaurene synthase and kaurene 24


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oxidase involved in gibberellin biosynthesis from Scoparia dulcis. J. Nat. Med.

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2018,72,456-463.

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(21) Studier, F.W.; Rosenberg, A.H.; Dunn, J.J.; Dubendorff, J.W. Use of

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T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol.

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1990,185,60-89.

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(22) Amann, E.; Ochs, B.; Abel, K.J. Tightly regulated tac promoter vectors

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useful for the expression of unfused and fused proteins in Escherichia coli.

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Gene 1988,69,301-315.

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(23) Guzman, L.M.; Belin, D.; Carson, M.J.; Beckwith, J. Tight regulation,

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modulation, and high-level expression by vectors containing the arabinose

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PBAD promoter. J. Bacteriol. 1995,177,4121-4130.

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(24) Balzer, S.; Kucharova, V.; Megerle, J.; Lale, R.; Brautaset, T.; Valla, S.

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A comparative analysis of the properties of regulated promoter systems

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commonly used for recombinant gene expression in Escherichia coli. Microb.

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Cell Fact. 2013,12,26.

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(25) Dvorak, P.; Chrast, L.; Nikel, P.I.; Fedr, R.; Soucek, K.; Sedlackova, M.;

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Chaloupkova, R.; de Lorenzo, V.; Prokop, Z.; Damborsky, J. Exacerbation of

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substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic

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metabolic pathway. Microb. Cell Fact. 2015,14,201.

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Yang,

X.W.;

Jian,

H.H.;

Wang,

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pSW2,

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Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous

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Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3. Appl.

25



553

Environ. Microbiol. 2015,81,5519-5526.

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(27) Fu, L.; Lu, C. A novel dual vector coexpressing PhiX174 lysis E gene

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and staphylococcal nuclease A gene on the basis of lambda promoter pR and

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pL, respectively. Mol.Biotechnol. 2013,54,436-444.

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(28) Chen, L.H.;Cai, F.; Zhang, D.J.; Zhang, L.; Zhu, P.; Gao, S.

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Large-scale purification and characterization of recombinant human stem cell

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factor in Escherichia coli. Biotechnol. Appl.Biochem. 2017,64,509-518.

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(29) Liu, Z.; Xu, C.; Zhang, J.; Chen, Y.; Liu, X.; Wu, L.; Zhang, Z.;Meng, X.;

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Liu, H.; Jiang, Z.; Wang, T. Functionally active rat S100A4 from a polymerase

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chain reaction-synthesized gene expressed in soluble form in Escherichia coli.

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Oncol.Lett. 2014,7,1179-1184.

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(30) Ding, D.; Liu, S.; Wang, K., Huang, L.; Zhao, J. Article expression,

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purification, and characterization of Cu/ZnSOD from Panax ginseng.

566

Molecules. 2014,19,8112-8123.

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(31) Zhu, W.; Yang, G.; Zhang, Y.; Yuan, J.; An, L. Generation of

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biotechnology-derived Flavobacterium column are ghosts by PhiX174 gene

569

E-mediated inactivation and the potential as vaccine candidates against

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infection in grass carp. J. Biomed.Biotechnol. 2012,2012,760730.

571

(32) Ge, X.; Yang, D.S.C.; Trabbic-Carlson, K.; Kim, B.;Chilkoti, A.; Filipe,

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C.D.M. Self-cleavable stimulus responsive tags for protein purification without

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chromatography. J. Am. Chem. Soc. 2005,127,11228-11229.

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(33) Banki, M.R.; Feng, L.A.; Wood, D.W. Simple bioseparations using 26


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575 576

self-cleaving elastin-like polypeptide tags. Nat Methods 2005,2,659-661. (34) Wu, W.Y.; Mee, C.; Califano, F.; Banki, R.; Wood, D.W. Recombinant

577

protein

purification

578

2006,1,2257-2262.

by

self-cleaving

aggregation

tag.

Nat.Protoc.

579

(35) Fong, B.A.; Wu, W.Y.; Wood, D.W. Optimization of ELP-intein

580

mediated protein purification by salt substitution. Protein Expr. Purif.

581

2009,66,198-202.

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(36) Shi, C.; Meng, Q.; Wood, D.W. A dual ELP-tagged split intein system

583

for non-chromatographic recombinant protein purification. Appl. Microbiol.

584

Biotechnol. 2013,97,829-835.

585

(37) Coolbaugh, M.J.; Shakalli, Tang. M.J.; Wood, D.W. High-throughput

586

purification

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recombinant

587

Anal.Biochem. 2017,516,65-74.

proteins

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self-cleaving

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tags.

588

(38) Freuler, F.; Stettler, T.; Meyerhofer, M.; Leder, L.; Mayr, L.M.

589

Development of a novel Gateway-based vector system for efficient,

590

multiparallel protein expression in Escherichia coli. Protein Expr. Purif.

591

2008,59,232-241.

592

(39) Ilari, A.; Fiorillo, A.; Angelaccio, S.; Florio, R.; Chiaraluce, R.; van der

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Oost, J.; Consalvi, V. (2009) Crystal structure of a family 16 endoglucanase

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from the hyperthermophile Pyrococcusfuriosus--structural basis of substrate

595

recognition. FEBS J. 2009,276,1048-1058.

596

(40) Bailey, M.J. A note on the use of dinitrosalicylic acid for determining 27



597

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598

1988,29,494-496.

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Biotechnol.

599

(41) Cheong, D.E.; Chang, W.S.; Kim, G.J. A cloning vector employing a

600

versatile β-glucosidase as an indicator for recombinant clones. Anal.Biochem.

601

2012,425,166-168.

602

(42) An, Y.; Meresse, P.; Mas, P.J.; Hart, D.J. CoESPRIT: a library-based

603

construct screening method for identification and expression of soluble protein

604

complexes. PLoS One 2011,6,e16261.

605 606 607 608 609 610 611 612 613 614 615 616 617 618 28


Page 29 of 43


619

Fig. 1 The pANY2 plasmid and its multiple cloning sites. (a) Map showing

620

the pANY2 plasmid and its multiple cloning sites. The ccdB cassette for toxic

621

CcdB protein expression is flanked by multiple cloning sites MCS-1 and

622

MCS-2. MCS-2 is followed by a hexahistidine tag (His-tag) site for protein

623

purification; (b) Cloning strategy employing two BsaI sites unidirectional

624

sticky-end cloning.

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 29



641

Fig. 2 Outline of the BsaI-based strategy for background free cloning. (a)

642

shows the DNA insert flanked by BsaI restriction sites from PCR and the vector

643

pANY2 are digested with BsaI, and ligated with T4 DNA ligase to generate the

644

plasmid; (b) The linearized pANY2 vector cannot perform intramolecular

645

self-ligation or intermolecular self-ligation because the sticky ends of pANY2

646

introduced by BsaI digestion do not arise from a palindromic site.

647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 30


Page 30 of 43

Page 31 of 43


663

Fig. 3 Procedures for preparing T-vector and performing unidirectional

664

TA cloning. The T-vector can be prepared by digestion with AhdI. The DNA

665

insert is amplified and 3′-A added by PCR using Taq DNA polymerase. Both

666

T-vector and DNA insert contain part of the recognition sequence of AvrII and

667

NcoI. Therefore, ligation products with the insert in the reverse orientation

668

regenerate the NcoI and AvrII sites, permitting elimination of this plasmid by

669

inclusion of AvrII and (or) NcoI enzymes in the ligation reaction. This ensures

670

unidirectional cloning.

671

672

673

674

675

676

677

678

679

680

31



Page 32 of 43

681

Fig. 4 Analysis of pfLamA expressed in E. coli BL21 (DE3) harboring

682

pET11-pfLamA,

683

SDS-PAGE gel shows crude and purified pfLamA from different strains. Lane

684

M: protein molecular weight marker; lanes 1, 3, 5, 7 show crude extracts from

685

E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA, pANY2-TA-pfLamA and

686

pANY2-sticky-pfLamA, respectively; lanes 2, 4, 6, 8 show Ni-NTA purified

687

proteins from E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA,

688

pANY2-TA-pfLamA and pANY2-sticky-pfLamA, respectively; (b) activity of

689

purified pfLamA from different strains assayed using the Congo red agar

690

method. Sectors 1-4 show E. coli BL21 (DE3) harboring pANY2,

691

pET11-pfLamA, pANY2-TA-pfLamA and pANY2-sticky-pfLamA, respectively;

692

(c) shows the activity of purified pfLamA from different strains assayed by DNS

693

assay. Tube 1 is the negative control of curdlan; tube 2-5 show curdlan

694

catalyzed by Ni-NTA purified proteins from E. coli BL21 (DE3) harboring

695

pANY2, pET11-pfLamA, pANY2-TA-pfLamA and

696

respectively.

pANY2-TA-pfLamA

and

pANY2-sticky-pfLamA.

697 698 699 700 701 702 32


(a)

pANY2-sticky-pfLamA,

Page 33 of 43


703

Fig. 5 Construction of pANY3 and pANY6 plasmids. (a) The pANY3

704

plasmid for temperature-induced expression. Elements pL and pR are

705

temperature sensitive promoters, and Cits857 encodes a temperature

706

sensitive repressor. The other regions are consistent with pANY2; (b)

707

screening for positive clones of pANY3-pfLamA via bacterial lysate

708

electrophoresis. Lanes M1 and M2, DNA ladders; lane 2, plasmid

709

pANY3-pfLamA extracted by plasmid DNA purification kit; lane 3, linearized

710

pANY3-pfLamA; lanes 3-12, bacterial lysate electrophoresis of the randomly

711

selected clones used to screen positive clones of the constructed

712

pANY3-pfLamA; (c) the pANY6 plasmid for ELP-intein-based protein

713

purification. ELP encodes ELP tag, and intein encodes self-cleaving intein. The

714

other regions are consistent with pANY2; (d) the strategy for ELP-intein-based

715

protein purification with pANY6. A target protein (TP) is expressed as part of a

716

fusion protein of ELP-intein-TP, which is extracted from cells with or without the

717

presence of urea. Then ELP-intein-TP is reversibly precipitated at high salt

718

concentrations, and the TP can be separated from ELP-intein via the

719

self-cleaving intein at low pH.

720

721

722

723 33



Page 34 of 43

724

Fig. 6 Temperature-induced expression of pfLamA and Hj-Xyn in E. coli

725

BL21 (DE3) harboring pANY3-pfLamA and pANY3-Hj-Xyn. (a) SDS-PAGE

726

gel shows crude and purified pfLamA and Hj-Xyn after temperature-induced

727

expression. Lane M: protein molecular weight marker; lanes 1 and 2 show

728

crude extract from E. coli BL21 (DE3) harboring pETM11 and purification of

729

this crude extract, respectively; lanes 3 and 4 show the crude extracts from E.

730

coli

731

temperature-induced protein expression; lanes 5 shows purification of pfLamA

732

from temperature-induced expression; lanes 6 and 7 show the crude extracts

733

from E. coli BL21 (DE3) harboring pANY3-Hj-Xyn before and after

734

temperature-induced protein expression; lane 8 shows purification of Hj-Xyn

735

from temperature-induced expression; (b) degradation of curdlan using

736

pfLamA from temperature-induced expression. Tube 1 shows curdlan without

737

enzyme treatment; tubes 2 and 3 show curdlan treated with crude and purified

738

pfLamA, respectively; (c) the activities of Hj-Xyn assayed by DNS assay. Tube

739

1 shows the negative control; tube 2 and 3 show xylan incubated with crude

740

and purified Hj-Xyn, respectively; (d) the expression levels of pfLamA and

741

Hj-Xyn under different induction temperatures.

BL21

(DE3)

harboring

pANY3-pfLamA

742

743

744 34


before

and

after

Page 35 of 43


745

Fig. 7 ELP-intein-based purification of partially soluble protein pfLamA

746

expressed from pANY6. (a) SDS-PAGE gel shows purification of pfLamA

747

using the ELP-intein-based method. Lane M: protein molecular weight marker;

748

lanes 1 and 2 show crude extract from E. coli BL21 (DE3) harboring

749

pETM11and pANY6-pfLamA, respectively; lanes 3-8 show the precipitated

750

ELP-intein-pfLamA after self-cleavage of intein at pH 8.5, 8.0, 7.5, 7.0, 6.5 and

751

6.0, respectively; lane 9 shows purified pfLamA; (b) and (c) show the effects of

752

(NH4)2SO4 and NaCl concentrations on the amount of precipitated protein,

753

respectively; (d) the phase transition temperature of the expressed fusion

754

protein ELP-intein-pfLamA; (e) the effect of the urea-based denaturation and

755

refolding processes on final concentration and total activity of the purified

756

pfLamA. C1 and C2 show final concentrations of purified pfLamA with and

757

without urea treatment, respectively; TA1 and TA2 show total activities of

758

purified pfLamA with and without urea treatment, respectively.

759 760 761 762 763 764 765 766

35



767

TOC:

768

36


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Fig. 1 The pANY2 plasmid and its multiple cloning sites. (a) Map showing the pANY2 plasmid and its multiple cloning sites. The ccdB cassette for toxic CcdB protein expression is flanked by multiple cloning sites MCS-1 and MCS-2. MCS-2 is followed by a hexahistidine tag (His-tag) site for protein purification; (b) Cloning strategy employing two BsaI sites unidirectional sticky-end cloning. 115x74mm (300 x 300 DPI)



Fig. 2 Outline of the BsaI-based strategy for background free cloning. (a) shows the DNA insert flanked by BsaI restriction sites from PCR and the vector pANY2 are digested with BsaI, and ligated with T4 DNA ligase to generate the plasmid; (b) The linearized pANY2 vector cannot perform intramolecular self-ligation or intermolecular self-ligation because the sticky ends of pANY2 introduced by BsaI digestion do not arise from a palindromic site. 99x116mm (600 x 600 DPI)


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Fig. 3 Procedures for preparing T-vector and performing unidirectional TA cloning. The T-vector can be prepared by digestion with AhdI. The DNA insert is amplified and 3′-A added by PCR using Taq DNA polymerase. Both T-vector and DNA insert contain part of the recognition sequence of AvrII and NcoI. Therefore, ligation products with the insert in the reverse orientation regenerate the NcoI and AvrII sites, permitting elimination of this plasmid by inclusion of AvrII and (or) NcoI enzymes in the ligation reaction. This ensures unidirectional cloning. 79x73mm (600 x 600 DPI)



Fig. 4 Analysis of pfLamA expressed in E. coli BL21 (DE3) harboring pET11-pfLamA, pANY2-TA-pfLamA and pANY2-sticky-pfLamA. (a) SDS-PAGE gel shows crude and purified pfLamA from different strains. Lane M: protein molecular weight marker; lanes 1, 3, 5, 7 show crude extracts from E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA, pANY2-TA-pfLamA and pANY2-sticky-pfLamA, respectively; lanes 2, 4, 6, 8 show NiNTA purified proteins from E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA, pANY2-TA-pfLamA and pANY2-sticky-pfLamA, respectively; (b) activity of purified pfLamA from different strains assayed using the Congo red agar method. Sectors 1-4 show E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA, pANY2-TApfLamA and pANY2-sticky-pfLamA, respectively; (c) shows the activity of purified pfLamA from different strains assayed by DNS. Tube 1 is the negative control of curdlan; tube 2-5 show curdlan catalyzed by NiNTA purified proteins from E. coli BL21 (DE3) harboring pANY2, pET11-pfLamA, pANY2-TA-pfLamA and pANY2-sticky-pfLamA, respectively. 56x38mm (600 x 600 DPI)


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Fig. 5 Construction of pANY3 and pANY6 plasmids. (a) The pANY3 plasmid for temperature-induced expression. Elements pL and pR are temperature sensitive promoters, and Cits857 encodes a temperature sensitive repressor. The other regions are consistent with pANY2; (b) screening for positive clones of pANY3pfLamA via bacterial lysate electrophoresis. Lanes M1 and M2, DNA ladders; lane 2, plasmid pANY3-pfLamA extracted by plasmid DNA purification kit; lane 3, linearized pANY3-pfLamA; lanes 3-12, bacterial lysate electrophoresis of the randomly selected clones used to screen positive clones of the constructed pANY3pfLamA; (c) the pANY6 plasmid for ELP-intein-based protein purification. ELP encodes ELP tag, and intein encodes self-cleaving intein. The other regions are consistent with pANY2; (d) the strategy for ELP-inteinbased protein purification with pANY6. A target protein (TP) is expressed as part of a fusion protein of ELPintein-TP, which is extracted from cells with or without the presence of urea. Then ELP-intein-TP is reversibly precipitated at high salt concentrations, and the TP can be separated from ELP-intein via the self-cleaving intein at low pH. 124x87mm (300 x 300 DPI)



Fig. 6 Temperature-induced expression of pfLamA and Hj-Xyn in E. coli BL21 (DE3) harboring pANY3pfLamA and pANY3-Hj-Xyn. (a) SDS-PAGE gel shows crude and purified pfLamA and Hj-Xyn after temperature-induced expression. Lane M: protein molecular weight marker; lanes 1 and 2 show crude extract from E. coli BL21 (DE3) harboring pETM11 and purification of this crude extract, respectively; lanes 3 and 4 show the crude extracts from E. coli BL21 (DE3) harboring pANY3-pfLamA before and after temperature-induced protein expression; lanes 5 shows purification of pfLamA from temperature-induced expression; lanes 6 and 7 show the crude extracts from E. coli BL21 (DE3) harboring pANY3-Hj-Xyn before and after temperature-induced protein expression; lane 8 shows purification of Hj-Xyn from temperatureinduced expression; (b) degradation of curdlan using pfLamA from temperature-induced expression. Tube 1 shows curdlan without enzyme treatment; tubes 2 and 3 show curdlan treated with crude and purified pfLamA, respectively; (c) the activities of Hj-Xyn assayed by DNS. Tube 1 shows the negative control; tube 2 and 3 show xylan incubated with crude and purified Hj-Xyn, respectively; (d) the expression levels of pfLamA and Hj-Xyn under different induction temperatures. 83x82mm (600 x 600 DPI)


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Fig. 7 ELP-intein-based purification of partially soluble protein pfLamA expressed from pANY6. (a) SDS-PAGE gel shows purification of pfLamA using the ELP-intein-based method. Lane M: protein molecular weight marker; lanes 1 and 2 show crude extract from E. coli BL21 (DE3) harboring pETM11and pANY6-pfLamA, respectively; lanes 3-8 show the precipitated ELP-intein-pfLamA after self-cleavage of intein at pH 8.5, 8.0, 7.5, 7.0, 6.5 and 6.0, respectively; lane 9 shows purified pfLamA; (b) and (c) show the effects of (NH4)2SO4 and NaCl concentrations on the amount of precipitated protein, respectively; (d) the phase transition temperature of the expressed fusion protein ELP-intein-pfLamA; (e) the effect of the urea-based denaturation and refolding processes on final concentration and total activity of the purified pfLamA. C1 and C2 show final concentrations of purified pfLamA with and without urea treatment, respectively; TA1 and TA2 show total activities of purified pfLamA with and without urea treatment, respectively. 116x75mm (300 x 300 DPI)


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