Supporting information
Genetically-modified
R-ω-transaminase:
purification
self-assembly facilitating interaction with substrate droplets
and
Qian Gao, Yaqi Fu, Yixiao Peng, Wei Liu, Wei Feng *
Department of Biochemical Engineering, Beijing University of Chemical Technology,
Beijing, 100029, China
Gene constructions and cloning for an elastin-like polypeptide (ELP)
A 20-repeat polypeptide of Val-Pro-Gly-Xaa-Gly was synthesized and cloned into
pUC57 plasmid by Genewiz Co. (Suzhou, China). (VPGXG)20 was used as the
monomer for synthesis of (VPGXG)100 and Xaa was chosen to be Val:Ala:Gly in a
5:3:2 ratio (Meyer and Chilkoti 2002).
pUC57-(VPGXG)20 was linearized with PflMI (2 μL PflM I; 3 μL 10×K buffer; 25
μL pUC57-(VPGXG)20 at 37 0C for 3 h), enzymatically dephosphorylated with
alkaline phosphatase, and then purified using a DNA extraction kit (Omega Bio-tek).
Another aliquot of the plasmid was codigested with PflMI and BglI restriction
endonucleases to generate the free (VPGXG)20 insert (2 μL PflM I; 2 μL Bgl I; 5 μL
10×K buffer; 41 μL pUC57-(VPGXG)20). After digestion, the reaction products were
separated by agarose gel electrophoresis, and the insert was purified using a DNA
extraction kit (Omega Bio-tek).
The monomers were then ligated to the linearized vector (0.4 μL T4 DNA ligase, 2
μL 10×ligation buffer; 2 μL pUC57-(VPGXG)20, 15.6 μL insert, incubated at 22 °C
for 20 min). A 10 μL portion of the ligation mixture was combined with 100 μL of
chemically competent Escherichia coli cells (DH5α), and the cells were transformed
* Corresponding author. Tel. +86-10-64446249. E-mail: fengwei@mail.buct.edu.cn;
by heat shock (30 min on ice, 90 s 42 °C, 3 min on ice). After addition of 900 μL LB
medium, the cells were cultured for 45 min, spread on LB medium agar plates
supplemented with ampicillin (50 μg/mL), and incubated at 37 °C. The transformants
were verified by their digestions with diagnostic restriction endonucleases and
confirmed by DNA sequencing (BGI Tech). The result of this process was a
(VPGXG)40 insert in the pUC-57 vector. Subsequent additional rounds of recursive
directional ligation proceed identically for (VPGXG)100. The plasmid pET28a was
co-digested BamHI and HindIII restriction endonucleases. The pUC-57 vector
harboring the (VPGXG)100 gene was co-digested BamHI and HindIII restriction
endonucleases, and the resulting fragment was ligated into the plasmid pET28a to
construct the expression vector pET28a/(VPGXG)100 in Escherichia coli.
Expression of ELP fusion D-amino acid oxidase. The expression vector
pET28a/(VPGXG)60 in Escherichia coli was constructed. The DAAO gene was
amplified by PCR using Pfu DNA polymerase with genomic DNA of Trigonopsis
variabilis as template using the paired primers. The DAAO gene was purified using a
DNA extraction kit (Omega Bio-tek) and was then co-digested with NcoI and BamHI
restriction endonucleases. The resulting fragment was ligated into the plasmid
pET28a/(VPGXG)60, which has been co-digested with NcoI and BamHI restriction
endonucleases. Thus the expression vector pET28a/(VPGXG)60-DAAO was
constructed in Escherichia coli.
Table 1. R-ω-transaminase gene sequence and primers
GCTAGCGTTGCGTTCTCTGCGGACACCTCTGAAATCGTTTACACCCACGACACCGGTCTGG
ACTACATCACCTACTCTGACTACGAACTGGACCCGGCGAACCCGCTGGCGGGTGGTGCGG
CGTGGATCGAAGGTGCGTTCGTTCCGCCGTCTGAAGCGCGTATCTCTATCTTCGACCAGGG
TTACCTGCACTCTGACGTTACCTACACCGTTTTCCACGTTTGGAACGGTAACGCGTTCCGTC
TGGACGACCACATCGAACGTCTGTTCTCTAACGCGGAATCTATGCGTATCATCCCGCCGCT
GACCCAGGACGAAGTTAAAGAAATCGCGCTGGAACTGGTTGCGAAAACCGAACTGCGTG
AAGCGTTCGTTTCTGTTTCTATCACCCGTGGTTACTCTTCTACCCCGGGTGAACGTGACATC
ACCAAACACCGTCCGCAGGTTTACATGTACGCGGTTCCGTACCAGTGGATCGTTCCGTTCG
ACCGTATCCGTGACGGTGTTCACGCGATGGTTGCGCAGTCTGTTCGTCGTACCCCGCGTTC
TTCTATCGACCCGCAGGTTAAAAACTTCCAGTGGGGTGACCTGATCCGTGCGGTTCAGGAA
ACCCACGACCGTGGTTTCGAAGCGCCGCTGCTGCTGGACGGTGACGGTCTGCTGGCGGAA
GGTTCTGGTTTCAACGTTGTTGTTATCAAAGACGGTGTTGTTCGTTCTCCGGGTCGTGCGGC
GCTGCCGGGTATCACCCGTAAAACCGTTCTGGAAATCGCGGAATCTCTGGGTCACGAAGC
GATCCTGGCGGACATCACCCTGGCGGAACTGCTGGACGCGGACGAAGTTCTGGGTTGCAC
CACCGCGGGTGGTGTTTGGCCGTTCGTTTCTGTTGACGGTAACCCGATCTCTGACGGTGTT
CCGGGTCCGATCACCCAGTCTATCATCCGTCGTTACTGGGAACTGAACGTTGAATCTTCTT
CTCTGCTGACCCCGGTTCAGTACGGATCC
The primers used for the amplification of the R-ω-transaminase gene.
ATGCTAGCGTTGCGTTCTCTGCGG
ATGGATCCGTACTGAACCGGGGTC
Table 2. ELP monomer
The gene sequence of the monomer (VPGXG)20 with a restriction site of SacI at 5'
terminal and a restriction site of SalI at 3' terminal
5'GGATCCGAGCTCCATATGGGCCACGGCGTGGGTGTTCCGGGCGTGGGTGTTCCGGGTGG
CGGTGTGCCGGGCGCAGGTGTTCCTGGTGTAGGTGTGCCGGGTGTTGGTGTGCCGGGTGTT
GGTGTACCAGGTGGCGGTGTTCCGGGTGCAGGCGTTCCGGGTGGCGGTGTGCCGGGCGTG
GGTGTTCCGGGCGTGGGTGTTCCGGGTGGCGGTGTGCCGGGCGCAGGTGTTCCTGGTGTA
GGTGTGCCGGGTGTTGGTGTGCCGGGTGTTGGTGTACCAGGTGGCGGTGTTCCGGGTGCA
GGCGTTCCGGGTGGCGGTGTGCCGGGCGGGCTGGTCGACAAGCTT3'
Table 3. DAAO gene sequence and primers
ATGGCTAAAATCGTTGTTATTGGTGCCGGTGTTGCCGGTTTAACTACAGCTCTTCAACTTCT
TCGTAAAGGACATGAGGTTACAATTGTGTCCGAGTTTACGCCCGGTGATCTTAGTATCGGA
TATACCTCGCCTTGGGCAGGTGCCAACTGGCTCACATTTTACGATGGAGGCAAGTTAGCCG
ACTACGATGCCGTCTCTTATCCTATCTTGCGAGAGCTGGCTCGAAGCAGCCCCGAGGCTGG
AATTCGACTCATCAACCAACGCTCCCATGTTCTCAAGCGTGATCTTCCTAAACTGGAAGGT
GCCATGTCGGCCATCTGTCAACGCAACCCCTGGTTCAAAAACACAGTCGATTCTTTCGAGA
TTATCGAGGACAGGTCCAGGATTGTCCACGATGATGTGGCTTATCTAGTCGAATTTGCTTC
CGTTTGTATCCACACCGGAGTCTACTTGAACTGGCTGATGTCCCAATGCTTATCGCTCGGC
GCCACGGTGGTTAAACGTCGAGTGAACCATATCAAGGATGCCAATTTACTACACTCCTCA
GGATCACGCCCCGACGTGATTGTCAACTGTAGTGGTCTCTTTGCCCGGTTCTTGGGAGGCG
TCGAGGACAAGAAGATGTACCCTATTCGAGGACAAGTCGTCCTTGTTCGAAACTCTCTTCC
TTTTATGGCCTCCTTTTCCAGCACTCCTGAAAAAGAAAATGAAGACGAAGCTCTATATATC
ATGACCCGATTCGATGGTACTTCTATCATTGGCGGTTGTTTCCAACCCAACAACTGGTCAT
CCGAACCCGATCCTTCTCTCACCCATCGAATCCTGTCTAGAGCCCTCGACCGATTCCCGGA
ACTGACCAAAGATGGCCCTCTTGACATTGTGCGCGAATGCGTTGGCCACCGTCCTGGTAGA
GAGGGCGGTCCCCGAGTAGAATTAGAGAAGATCCCCGGCGTTGGCTTTGTTGTCCATAAC
TATGGTGCCGCCGGTGCTGGTTACCAGTCCTCTTACGGCATGGCTGATGAAGCTGTTTCTT
ACGTCGAAAGAGCTCTTACTCGTCCAAACCTT
The primers used for the amplification of the DAAO gene, which were designed
based on the gene sequence of the DAAO (GenBank accession AY514426).
5'GCGCCATGGCTAAAATCGTTGTTATTG3'
5'ATGGATCCAAGGTTTGGACGAGTAAGAG3'
Fig. 1. Construction of the plasmid pET28a/(VPGXG)100/R-ω-TA in Escherichia coli
Fig. 2. Schematic presentation for the purification of ELP-R-ω-TA
Addition of NaCl (3M) to cell lysate with an ELP fusion R-ω-TA at 30 0C triggers the phase transition,
causing ELP-R-ω-TA to aggregate. Centrifugation separates the aggregated ELP-R-ω-TA from other
contaminants. The aggregated ELP-R-ω-TA in the pellet after centrifugation was dissolved in cold PBS
(4 °C) followed by centrifugation at 4°C. The cold spin removed insoluble contaminants that were
trapped with the aggregated ELP-R-ω-TA.
Fig. 3. Conversion of (R)-1-phenylethylamine (30 mM) catalyzed by
ELP-R-ω-TA and R-ω-TA. The concentration of amine acceptor pyruvate is 30 mM.
a
b
Fig. 4. Analysis of ELP-DAAO by SDS-PAGEA (a) and confocal image of
ELP-DAAO (b)
Lane M: molecular mass marker (KDa); lane 1 is for the supernatant from the third round of inverse
transition cycling; lane 2 is for total proteins.