Single Protein Production (SPP)
The SPP System consists of two plasmids that are co-transformed for expression. One vector carries the E. coli toxin protein, mazF, and the other is a modified pCold vector that does not contain any ACA sequences. As previously stated, the mazF protein was determined to be a sequence-specific endoribonuclease that cleaves single-stranded RNAs at ACA sequences. In order to effectively use this system, the transcript of interest should not contain any ACA sequences; i.e., should be ACA-less.
The ACA-less gene of interest can be constructed by chemical synthesis or site-directed mutagenesis technology. In designing the ACA-less gene, every ACA sequence within the gene of interest must be modified to make its amino acid sequence and restriction sites for cloning in frame. This ACA-less gene is then cloned into pCold (SP-4) vectors, which are ACA-less pCold vectors.
An E. coli host is then co-transformed with this expression plasmid and the pMazF expression plasmid. Expression of mazF is induced, cleaving all other cellular mRNA derived from the host proteins or others at ACA sequences but leaving the target mRNA intact. The result is highly pure (up to 90% of cellular protein) target protein expression with almost no cellular protein background. Despite the stress of undergoing a drop in growth temperature, target protein synthesis can be detected for up to four days.(3,4) Figure 2 illustrates the design of the SPP System.
To demonstrate the viability of the system for structural analysis studies, an experiment using the envZB gene was performed. A control expression plasmid was prepared by inserting an ACA-less ORF of E. coli-derived envZB protein into pCold I (SP-4) DNA. The estimated molecular weight of the expressed protein is 19.6 kDa. E. coli BL21 cells were co-transformed with pCold I (SP-4) vector and pMazF in 3 mL of M9-glucose medium with 50 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37C.
Once the OD600 reached 0.5, the culture was placed at 15C for 45 minutes in order to facilitate cold shock expression. mazF and envZB induction was initiated with the addition of 1 mM IPTG. After 24 hours of induction, 250 µL of the culture was added to 19 amino acids (50 µg/mL each, minus methionine) and 15µCi [35S]-methionine. At the end of a 15-minute isotopic labeling at cold shock temperatures, nonradioactive methionine was added and incubated for an additional five minutes.
Labeled cells were washed in a Tris-Cl, NaCl buffer and then centrifuged to remove the buffer. The pellets were resuspended in SDS-PAGE loading buffer and resolved on 15% SDS-polyacrylamide gel. After drying, the gel was exposed to X-ray film overnight. Results are shown in Figure 3.
The pulse labeling results demonstrate that using the SPP System, a union of pCold vectors and mazF, up to 90% of the newly synthesized protein expressed is the protein of interest. mazF degraded host proteins mRNAs, resulting in low cellular protein background and enhancement of the ACA-less target protein.