Co-expression of several target genes in E. coli is advantageous for studying protein complexes; the expression of protein complexes can also sometimes help to increase the solubility of expressed protein in E. coli. However, there are some obstacles to co-expression, such as different replication origin requirements and different antibiotic resistance markers for selection. Here, the Duet™ Expression System from Novagen (part of Merck4Bioscoences) satisfies all the requirements for multiple target gene expression.
Each Duet vector has compatible replicons and antibiotic resistance markers as shown in the example below. All of the Duet vectors have T7 promoters for expression of target genes by IPTG induction; each Duet vector is designed to co-express two target proteins in E. coli. Novagen provides 5 different types of Duet vectors. The compatible replicons and antibiotic resistance genes are also designed to allow for effective propagation and maintenance of four different plasmids in a single E. coli cell. pETDuet-1 carries the ColE1 replicon and bla gene for ampicillin resistance, pACYCDuet-1 carries the P15A replicon and cat gene for chloramphenicol resistance, and pCDFDuet-1 carries the CloDF13 replicon and aadA gene for streptomycin/spectinomycin resistance. Two kanamycin-resistant Duet vectors are available: pRSFDuet-1 carries the RSF1030 replicon and pCOLADuet-1 carries the ColA replicon. Each vector carries two expression units each controlled by a T7 lac promoter for high-level protein expression. By combination of 4 of those Duet vectors, 8 different proteins could be expressed in a single E. colicell.
For a better understanding of the Duet system, let’s look at one of them. pETDuet-1 is designed for the co-expression of two target genes; this vector includes two multiple cloning sites (MCS). Each MCS has a T7 promoter, lac operator and ribosome binding site for the expression of the target gene. The promoter is followed by the ribosome binding site and MCS. The pETDuet-1 vector can be transformed into the same cell with pCDFDuet-1, pACYCDuet-1, pRSFDuet-1 and/or pCOLADuet-1 in the appropriate host strain for the co-expression of up to 8 target genes. However, host strain compatibility should be considered for good expression.
Genes inserted into MCS-1 can be sequenced using the pETUpstream primer and DuetDOWN primer. Genes inserted into MCS-2 can be sequenced by using the DuetUP2 primer and T7 terminator primer. Each Duet vector also provides His•Tag® and S•Tag™ sequences for detection and purification of protein complexes. Each tag can be removed by adding a stop codon at the end of the target gene sequence in order to get an un-fused native protein.
One of my target proteins had very low solubility and I had previously experienced difficulties purifying it using his-tag. But by adding a known binding partner in pETDuet-1, I could co-express the two proteins and perform the purification by increasing the solubility of my target protein. In my experiment, my target protein had the his-tag for the purification but the binding partner did not have any tag. By using Ni-NTA, I co-purified the binding partner and target protein as they made a complex in E. coli. Please note that although I only expressed two different proteins in my experiment, theoretically, we could express 8 different proteins in this system.