Thermostability and Molecular Encapsulation within an
Engineered Caged Protein Scaffold
Mercè Dalmau, Sierin Lim, Helen C. Chen, Cesar Ruiz, and Szu-Wen Wang
Department of Chemical Engineering and Materials Science
University of California
Irvine, CA 92697-2575
SUPPLEMENTARY MATERIAL
Gene synthesis.
The DNA sequence of the E2 wild-type protein scaffold
was optimized by CODA Genomics (Laguna Hills, CA). In this algorithm, both synthetic
gene assembly and expression in the heterologous organism (E. coli) are optimized by
determining favorable oligonucleotide hybridization temperatures and codon usage,
respectively (Hatfield and Roth 2007; Lathrop and Hatfield 2007).
We assembled
twenty-five oligonucleotides encoding the E2-WT gene into four intermediate fragments
using PCR cycling with PfuUltra High-Fidelity DNA polymerase and CODA Blue
primer mix (CODA Genomics). Each fragment was then cloned into the vector CODA
Blue. These four intermediate segments were then PCR amplified, and equal amounts of
each intermediate PCR product were mixed and subjected to another round of PCR
extension and amplification. The resulting full-length gene was digested with HindIII,
ligated into the CODA Blue vector, and sequenced.
Construction of plasmid. To incorporate a stop codon (TAA) and NdeI and
BamHI restriction endonuclease sites for cloning, the synthetic gene was PCR-amplified
using PfuUltra High-Fidelity DNA polymerase from the assembly vector with primers
5’-gggaattccatATGCTGTCTGTTCCTGGTCCC–3’
(forward)
5’-cgcggatccTTAAGCTTCCATCAGCAGCAG–3’ (reverse).
and
The E2-WT DNA
sequence is denoted in capital letters and the NdeI and BamHI sites are underlined.
To construct the expression vector pE2 containing the wild-type scaffold gene, we
digested both the vector pET-11a (Novagen) and the amplified synthetic gene with NdeI
and BamHI. The digested vector was treated with calf intestinal alkaline phosphatase and
both vector and amplified synthetic gene were purified by agarose gel electrophoresis.
We constructed the plasmid by ligating the vector backbone and synthetic gene with T4
DNA ligase. Selection of the construct was performed by transforming the ligation
mixture into DH5 (E. coli) grown on LB plates containing ampicillin.
DNA
manipulations were performed according to standard procedures (Sambrook and Russell
2001). The final expression plasmid (pE2) containing the optimized, wild-type scaffold
is shown in Figure S1.
Figure S1. Plasmid map of pE2 expression vector. “E2-WT” encodes the
synthetic E2 gene which has been truncated down to its core scaffold.
Bam HI (320)
Ap(R)
E2-WT
Nde I (1107)
lac operator
pE2
ORI
T7 promoter
6424 bp
lacI
Construction of mutants. To perform site-directed mutagenesis, we cloned the
E2-WT gene into pGEM-3Z (Promega) and followed a modified version of the
Stratagene QuickChange protocol. We used oligonucleotides with the sequence 5’–
ACCAAACTGGTTGCGCACCGTNNNAAATTCAAGGCGATTGCGGCG-3’
and
5’-GCCGAAAAGCCGATCGTTCGTNNNGGTGAAATCGTTGCTGCTCCG–3’
for
mutants at positions 239 and 381, respectively. NNN indicates the 239 or 381 mutation
site and was replaced with GCG, TTT, or TGC for Ala, Phe, or Cys, respectively. For
constructing the double-Phe mutant, site-directed mutagenesis was performed
consecutively. The resulting clones were screened and the mutated E2 genes were cloned
into the expression vector pET-11a at the NdeI and BamHI sites as described above for
the wild-type gene. The sequences of the mutant and wild-type genes were confirmed by
DNA sequencing.