pBAD102 and pBAD202 Directional TOPO® Expression Kits From Invitrogen

This is probably one of the best systems (if not the best) we have ever used in the laboratory to express heterologous recombinant proteins in bacteria! There are two pBAD directional cloning vectors available (102D- ampicillin resistance and 202D-kanamycin resistance). We have used pBAD102D to successfully express hepatocyte, Plasmodium parasite and mosquito proteins in TOP10 (One Shot) or BL21 competent cells. Blunt-end cloning of the gene of interest into the expression vector requires the synthesis of a new forward primer which includes a 5’-CACC overhang. This overhang allows for in-frame cloning and the expression of a fusion protein with N-terminal and C-terminal domains. The entire cloning step is quick: 5 minutes at room temperature or 30 minutes to 1 hr at room temperature for larger inserts. The vector is approximately 4.5 kb. We have used inserts from 400 bp to 1.9 kb and as such, do not know of any size limitations beyond this range. The sequencing primers that come with the kit are horrible (i.e. no reads or very short reads) and it is advisable that additional primers be designed to aid in sequencing and screening for clones with inserts. The inserts can only go in one-way (hence the directionality) so assuming that you have checked for PCR artifacts that can arise from incorrect primer design and low-fidelity polymerase activity, the positive clones should all express your protein of interest. We often observe only a few colonies (10-30) on the plate for any one experiment and we consider this to be “normal”. You only need one to work!

Regulation of protein expression by the araBAD promoter is tightly controlled by the AraC gene product. A simple pilot expression experiment can be conducted to determine the optimal concentration of L-arabinose to use for induction. The N-terminal leader domain contains a thioredoxin His-patch site. Thioredoxin sequence increases the solubility of the expressed protein while the His-patch aids in subsequent purification by nickel chromatography. The leader domain contains an enterokinase cleavage site that allows for excision of the N-terminus from the expressed protein. The C-terminus encodes for a 6x-His domain for nickel purification with an additional V5 epitope for detection. Anti-His, Anti-Thio and anti-V5 antibodies work equally well in detecting proteins that are expressed correctly. We have used this vector to express secreted and membrane-bound proteins and found that this system is much faster, requires fewer intervening steps and is generally easier to use than the pET vector that we have in the laboratory. By and large, the majority of postdocs in the lab have moved to the pBAD system for these very reasons. The pBAD system dovetails beautifully with the Takara Chaperone Plasmid system. Read a review of the Takara Chaperone Plasmid System. To use this system, you will first need to transfect your pBAD clone into a pre-transformed BL21 cell containing the Takara Chaperone Plasmid. The Takara plasmid facilitates the expression of heterologous proteins by over-expressing bacterial chaperones. The expression of proteins from both plasmids is induced by the addition of L-arabinose. The use of this latter system is especially useful for problematic insoluble proteins.

The pBAD Directional TOPO® Expression Kits are not cheap. However, if you calculate the time and effort required to express proteins using other vectors with the “one shot deal” of this system, then it is well worth the investment!