The following constructs were generated in the pSB1Ak8 vector:
T7 promoter-RBS-RiAFP-His-Terminator
T7 promoter-RBS-eGFP-ZeAFP-His-Terminator
T7 promoter-RBS-His-eGFP-TEV-TmAFP-Terminator
T7 promoter-RBS-His-eGFP-TmAFP-Terminator
In the pSB1A3 vector:
A plasmid containing the TmAFP antifreeze protein was kindly provided by the Fass lab. A plasmid containing the type III ZeAFP antifreeze protein was kindly provided by the Davies lab. The DNA sequence encoding the 12kDa RiAFP was codon-optimized for E. coli, synthesized, and generously sponsored by Integrated DNA Technologies.
All genes were PCR-amplified with primers of the following form:All primer designs can be found in the protocols section.
PCR products were PCR purified and gel purified, then cut with XbaI and PstI and ligated to the pSB1AK8 plasmid (containing a T7 promoter) cut with SpeI and PstI. Successful ligation was verified by colony PCR, a double digest, and sequencing. Several antifreeze constructs were transferred from the pSB1AK8 vector to the pSB1C3 vector for submission to the registry. The pSB1C3 linearized vector and the biobricks in the pSB1AK8 vector were digested with EcoRI and PstI. The pSB1AK8 vector was CIP treated prior to ligation. Colonies were selected on chloramphenicol plates, and colony PCR and sequencing were used to verify the constructs.
all of the dna figures are in this div
Expression and Purification All constructs were over-expressed in either the BL21*(DE3) strain or the Origami 2 (DE3) plyS strain. An aliquot of the origami strain was kindly provided by the Xiong Lab. This strain has mutations in both the thioredoxin reductase and glutathione reductase genes, which greatly enhance disulfide bond formation in the E. coli cytoplasm. Cell cultures were grown to OD ~0.5 and then induced by addition of 0.5mM-1mM IPTG. Several temperatures and length of induction were investigated to optimize yields. TmAFP cultures were lowered to 16oC and shaken for a further 40-48 hours. RiAFP cultures were lowered to 22oC and shaken overnight. Cells were spun down at 4700rpm for 20 minutes. Pellets were resupended in lysis buffer, sonicated, and centrifuged once more. Protein samples were electrophoresed on a gradient 4% to 20% SDS-PAGE. Gels were either stained with Coomassie blue or were transblotted onto nitrocellulose membrane with Invitrogen iBlot Dry Blotting. Western blotting was performed according to manufacturer’s protocol, using mouse α-GFP-IgG2a antibody and rabbit α-his antibody (Santa Cruz). More details for this and all other protocols can be found in our “protocols” section. The HisTrapTM purification column was used to purify the RiAFP recombinant protein. The cobalt agarose beads of the column were equilibrated with lysis buffer. Lysed samples were filtered through a 0.22uM filter and passed through the column. Fractions were eluted with an imidazole gradient. Fractions were over-loaded on a gel to check for purity, and pure fractions were combined and concentrated using a 10K molecular weight cutoff filter. For the purification of RiAFP, fusion protein samples were incubated overnight with TEV protease. Size exclusion chromatography was used to isolate RiAFP from the fusion protein. For crystallography purposes, size exclusion chromatography was used to obtain exceptional purity of RiAFP and RiGFP samples. Concentrations of protein were measured using a nanodrop A280, a Bradford Assay, and/or UV-vis spectroscopy. All samples were successfully over-expressed in both BL21 and Origami cells. Expression was readily detected by SDS-PAGE and/or Western blotting. Some expression was also noted in the uninduced sample; this likely resulted from leaky expression of the T7 RNA polymerase gene and is a normal occurrence for the BL21 strain. No toxicity effects were observed due to recombinant expression. Strains expressing GFP-fused RiAFP formed bright green pellets after centrifugation post-induction. Importantly, GFP-fused RiAFP was produced at concentrations of approximately 0.2 millimolar (approximately 150mg/mL). This is several orders of magnitude greater than the expression levels achieved with TmAFP, which were in the micromolar range. Most of the TmAFP was observed in an insoluble pellet fraction (verified by SDS-PAG). The nanodrop instrument was not sensitive enough to use UV-vis spectroscopy to determine the concentration of soluble protein; instead we used flourimetry to obtain a rough estimate. Flourometric measurements were recorded using Photon Technology International Flurometer. An excitation wavelength of 488nm and an emission scan from 500nm to 650nm were used to measure fluorescence. The fact that we were able to achieve the first ever large-scale recombinant production of an insect antifreeze protein is significant. Inability to produce insect antifreeze proteins in large quantities without the use of expensive refolding protocols has been a limiting factor for their use in industry. We believe that RiAFP, which already has one of the highest thermal hysteresis activities of all known antifreeze proteins, is thus an attractive reagent to be used in industrial applications requiring freeze resistance.
more pics for protein work
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