Download pSABAD92A

pSABAD92A
pSKDuet12 was constructed by inversion PCR of pSKDuet1A [1] using oligonucleotides
#SK165
(CACCGTAACGGAGACAAAATGTCTAAGCTATG)
and
#SK160
(TGTCTCCGTTACGGTGTAGGTTTTGG) and carries a fusion protein with an N-terminal
His-Tag, the, B1-domain of G-protein (GB1) and the N-terminal fragment (IntN) of the native
split NpuDnaE intein[2]. The primers #DuetMCS-fw (GGATCTCGACGCTCTCCCT) and
#HK010 (CGCATCTCGAGTTCGGCAAATTATCAAC) were used to amplify part of
pSKDuet12 with PCR. The PCR product was then ligated into pCDFDuet-1 (plasmid 15917
from Addgene) using the NcoI and XhoI sites resulting in pTMCDF01. An His-Tag was
inserted
by
ligating
the
oligonucleotide
#SZ008
(TCGACTCATCATCATCATCATCATTAA)
and
#SZ009
(TCGACTTAATGATGATGATGATGATGA) into pTMCDF01 using newly created XhoI
site to result in plasmid pTMCDF02. Furthermore, an EcoRI site and an EF-linker (Glu-Phe)
was
created
by
inversion
PCR
using
oligonucleotides
#HK038
(GATAATTTGCCGAACGAATTCCATCATCATCATC)
and
#HK039
(GATGATGATGATGGAATTCGTT) to end up into pHKCDF22-3. Finally the primers
#SK012
(TCCTTACATATGCAGTACAAACTTATC)
and
#HK122
(CTAAAGCTTAATGATGATGATGATGATG) were used to amplify a part of
pHKCDF22-3 and the PCR product was ligated between the NdeI and HindIII sites of
pSKBAD2A [1] (plasmid 15335 from Addgene) vector to yield pMHBAD10. To gain the
fusion-protein IntC- B1-domain of G-protein (GB1)-His-Tag, a part of pSKBAD2A was
amplified with oligonucleotides #SK094 (TAACATATGATCAAAATAGCCACACG) and
#HK158 (AGAATTCCGTTACGGTGTAGGTTTTG), and ligated into pMHBAD10
between the NdeI and EcoRI sites resulting in pMHBAD14. Then GB1 was replaced by the
R-module of AlgE4.
To obtain the R-module template, pSKDuet1A was digested with NcoI and HindIII and
ligated into pRSF-1b (Novagen) resulting in pHYRSF1. pSABAD28 was constructed by
PCR using pSKBAD2A as a template and using oligonucleotides #SK094
(TAACATATGATCAAAATAGCCACACG),
#HK036
(CCGCGGGCGTTCGTGCAATTAGAAGCTATGAAGCC),
and
#HK037
(CAGGTACCGCCAGCCCCGCGGGCGTTCGTGC) and ligating the product into
pSKBAD2A between the NdeI and KpnI sites. pSABAD28 was digested with NdeI and
HindIII and the digestion product was ligated into pHYRSF1 obtaining pSARSF1-28.
pSARSF1-66 was constructed by inversion PCR of pSARSF1-28 using oligonucleotides
#HK152
(GACGCTGCTACCGCCGAAAAAGTTTTCAAAC)
and
#HK153
(GTTTGAAAACTTTTTCGGCGGTAGCAGCGTC). Finally pSARSF1-LICI-1 was
constructed by ligation independent cloning (LIC) by amplifying the part of pFA1 [3], that
contains the gene of the R-module of AlgE4, with oligonucleotides #HK137
(GCACGAACGCCCAAGGAAGCGACGGCGAGCCAC)
and
#HK138
(ACCGCCAGCCCCTTAGACGATCGCCCCGGCCTG) and inserting the ligation product
into pSARSF1-66 using the SacII site. The gene of the R-module of AlgE4 was amplified
from
pEBBAD30A
with
oligonucleotides
#HK057
(AAGGTACCGGAAGCGACGGCGAGCCAC)
and#HK197
(GTCTTCGCCGCGACCGAATTCA) and inserted it into pMHBAD14 between the KpnI
and EcoRI sites. pEBBAD30A was obtained by ligating the product from NcoI and HindIII
digestion of pSARSF1LICI-1f into pSKBAD2A. The resulting plasmid pSABAD92 encodes
a fusion protein consisting of the C-terminal 36 residues (IntC) of the NpuDnaE intein from
Nostoc punctiforme,the AlgE4 R-module and the C-terminal His-Tag with an EF linker (GluPhe).
pEBDuet23A
The DNA fragment encoding the A-module (residues 1-379) of AlgE4 was amplified from
pBS32 by the two oligonucleotides # HK54 (CCTACCTGAAAAGTTTCGAGGCGGATGC)
and #HK141 (CCGGCGAACCCGGCGCGACAA) and cloned into pSKDuet12 using the
NcoI and AhdI sites, resulting in the plasmid pEBDuet23A.
pBS32 is a derivative of pTYB4 (NEB) in which a 1.65 kb NcoI-XmaI DNA fragment
corresponding to the full-length AlgE4 from pHH4 [4] was subcloned.
pEBDuet28A:
pTMDuet03 was constructed by cloning the N-terminal domain of the ClpX zinc finger from
the chromosomal DNA of E. coli DH5α using oligonucleotides #HK001
(TAGACCATGGCAGATAAACGCAAAGATG)
and
#HK002
(TTTCACGATGCGGTGCAAC), ligating the PCR product into pSKDuet12 using the NcoI
and AhdI sites. The N-terminal His-Tag and TEV-digestion site was created by inversion
PCR
using
oligonucleotides
#HK014
(CATGCGGGGTTCTCATCATCATCATCATCATGAGAATTTGTATTTTCAGTCCATG)
and
#HK015
(ATGGACTGAAAATACAAATTCTCATGATGATGATGATGATGAGAACCCCG).
pEBDuet23A was digested with HindIII and NcoI. The gene encoding the N-terminal
precursor A-IntN was inserted into pTMDuet03 to yield pEBDuet28A.
Figure S1:
A)
The plasmid pSABAD92A encodes a fusion protein consisting of the C-terminal 36 residues
(IntC) of the DnaE intein from Nostoc punctiforme and the AlgE4 R-module (residue 385533) of Azotobacter vinelandii. The last 20 residues of the wild type R-module were
exchanged to a C-terminal His-tag for purification. The construct pSABAD92A has a ColE1
origin for replication, the arabinose promoter and has also the ampilicin resistance gene
(ampR)
B)
The plasmid pEBDuet23A encodes a fusion protein consisting of the A-module (residues 1379) of the alginate epimerase AlgE4 and the N-terminal part (IntN) of the NpuDnaE intein .
The vector has also the kanamycin resistance gene (kanR), RSF origin and the expression of
the fusion gene is tightly controlled by T7/lac promoter.
C)
pEBDuet28A has also the kanamycin resistance gene (kanR), RSF origin and the T7/lac
promoter. A fusion protein consisting of a N-terminal His-Tag, the A-module of AlgE4 and
the N-terminal part (IntN) of the NpuDnaE intein.
References
1. Iwaï H, Züger S, Jin J, Tam PH (2006) Highly efficient protein trans-splicing by a
naturally split DnaE intein from Nostoc punctiforme. FEBS Lett 580: 1853-1858.
2. Caspi J, Amitai G, Belenkiy O, Pietrokovski S (2003) Distribution of split DnaE inteins in
cyanobacteria. Mol Microbiol 50: 1569-1577.
3. Aachmann FL, Svanem BG, Valla S, Petersen SB, Wimmer R (2005) NMR assignment of
the R-module from the Azotobacter vinelandii Mannuronan C5-epimerase AlgE4. J
Biomol NMR 31: 259.
4. Ertesvåg H, Høidal HK, Hals IK, Rian A, Doseth B, et al. (1995) A family of modular type
mannuronan C-5-epimerase genes controls alginate structure in Azotobacter
vinelandii. Mol Microbiol 16: 719-731.