This protocol provides a highly effective bacterial display system for isolation of novel variants of BirA that allows for specific biotinylation of native proteins. The main advantage of the technique is that it selects BirA variants using high-affinity streptavidin selection. This ensures that inactive clones are removed, thus creating a highly efficient selection process.
To begin, in a plasmid editor, design the forward primer to include the last 30 nucleotides of the reverse complement of the peptide coding sequence, and add the plasmid binding nucleotide sequence to its five-prime end. Design the reverse primer to include the first 30 nucleotides of the reverse complement of the peptide coding sequence, and add the plasmid binding nucleotide sequence to its five-prime end. Then, to set up a 20-microliter PCR reaction, add the reagents in a thin-walled PCR tube.
Transfer the tube to a thermal cycler, and perform PCR according to the manuscript. Next, run five microliters of the PCR reaction on a 1%agarose gel to confirm the amplification of a six-kilobase PCR product, and proceed according to the manuscript. Now, to synthesize the mutant megaprimers with BirA hexahistidine forward and reverse primers, in a PCR tube, add one nanogram of pBAD-BirA-eCPX with the prepared target peptide sequence as a template.
Set up 35 PCR cycles with an annealing temperature of 60 degrees Celsius according to the manufacturer's instruction. Then, run five microliters of the amplification reaction on a 1%agarose gel to verify the amplification of a 984-base pair PCR product. Purify the PCR product from the remaining 45 microliters of the amplification reaction using a commercial PCR purification kit, and use a spectrophotometer to quantitate the DNA yield.
In a thin-walled PCR tube, add the reagents in the prepared mutant megaprimers to prepare the sample reaction. Transfer the reaction mixture to a thermal cycler, and run the PCR according to the manuscript. Store the amplification reaction at four degrees Celsius or on ice.
Next, add one microliter of Dpn1 restriction enzyme directly to the amplification reaction, and mix gently. Spin down the reaction mixture, and incubate for two hours at 37 degrees Celsius. After two hours, transform T7 Express lysY/Iq competent E.coli cells in a tube with two microliters of the Dpn1 reaction.
First, inoculate 100 milliliters of LB containing 1%glucose and 100 micrograms per milliliter of ampicillin with one milliliter of BirA library, and incubate overnight at 37 degrees Celsius with shaking at 200 rpm. Then, inoculate five milliliters of LB containing 1%glucose and 100 micrograms per milliliter of ampicillin with 100 microliters of the overnight culture. Incubate for two hours and 30 minutes or until the culture reaches an OD600 of approximately 0.5.
Next, induce eCPX and BirA expression with 0.2%weight by volume L-arabinose, 100-micromolar IPTG, and 100-micromolar biotin. Shake the culture at 200 rpm for one hour at 37 degrees Celsius. Centrifuge the culture for 10 minutes at 5, 000 times g, and remove the supernatant.
Resuspend the cells in one milliliter of ice-cold PBS, and centrifuge at 5, 000 times g for five minutes. Discard the supernatant, and resuspend the cells in 400 microliters of ice-cold PBS, and store cells on ice. Then, transfer 10 microliters of the resuspended cells into a 1.5-milliliter tube labeled input.
Store on ice. Next, wash 20 microliters of streptavidin magnetic beads in one milliliter of ice-cold PBS in a tube, and place the tube in a benchtop magnetic particle separator for two minutes, and carefully remove the supernatant. Resuspend the streptavidin magnetic beads in 20 microliters of ice-cold PBS, and transfer the bead solution to the previous prepared 390 microliters of resuspended cells.
Mix by gently pipetting. Then, incubate for 30 minutes at four degrees Celsius. Place a column with ferromagnetic spheres in a magnetic particle separator, and wash with five milliliters of ice-cold PBS.
Transfer the cells and streptavidin magnetic beads from the tube into the column attached in the separator. Once the column reservoir is empty, wash the column with a total volume of five milliliters of ice-cold PBS at 500 microliters each time. After that, remove the column from the separator, and place it in a 1.5-milliliter tube.
Pipette one milliliter of the ice-cold PBS onto the column, and use the plunger to elute the magnetically labeled cells. Then, place the 1.5-milliliter tube into a benchtop separator, and wash the magnetically labeled cell with one milliliter of ice-cold PBS. Gently resuspend the magnetically labeled cells in one milliliter of the ice-cold PBS.
Transfer 10 microliters of the resuspension into a 1.5-milliliter tube labeled output. Inoculate 100 milliliters of LB containing 1%glucose and 100 micrograms per milliliter ampicillin with the magnetically labeled cells, and incubate overnight at 37 degrees Celsius with shaking at 200 rpm. The next day, combine 10 milliliters of the overnight culture in LB with glycerol to a final concentration of 15%and store at minus 80 degrees Celsius to make freezer stocks.
Use 100 microliters of the overnight culture for the next round of selection. Now, add 990 microliters of ice-cold PBS to both the input and output samples, and label the tubes input 10 to the minus two and output 10 to the minus two, respectively. Make 10-fold serial dilutions of the two samples in ice-cold PBS until a final dilution of 10 to the minus 10 is reached in the input sample and 10 to the minus four in the output sample.
Plate 100 microliters of samples from input 10 to the minus six, input 10 to the minus eight, input 10 to the minus 10, output 10 to the minus two, output 10 to the minus three, and output 10 to the minus four on individual LB-ampicillin plates, and incubate overnight at 37 degrees Celsius. In the morning, count the number of colonies on the plates with clearly separated colonies. Multiply the colony count with the dilution factor to obtain the bacterial concentration count per 100 microliters.
In this protocol, after generation of a randomly mutated library of BirA variants, BirA and eCPX-AP expression was induced. Bacteria were incubated with affinity reagent, unbound bacteria were discarded, and selected bacteria were amplified. Western blot of pBAD-BirA-eCPX-AP expressing bacteria produced a 22-kilodalton streptavidin-reacting band consistent with the molecular weight of eCPX in both uninduced and induced cultures.
However, they were not present in BirA hexahistidine. The strong surface biotinylation in the eCPX-AP expressing bacteria caused aggregation upon addition of streptavidin magnetic beads and the formation of a pellet at the bottom of the tube, which was not observed with eCPX with lysine to alanine mutation expression bacteria. Analysis of the precipitate from the streptavidin pulldown displayed a clear 22-kilodalton streptavidin-reacting and anti-hexahistidine band in the samples from eCPX-AP cultures but not eCPX-AP with lysine to alanine mutation cultures.
Similarly, the count of bacteria bound to the streptavidin beads was significantly higher in the eCPX-AP than the eCPX-AP lysine to alanine mutation cultures. The most important step is a stringent washing of streptavidin-bound bacteria. A stringent washing assures that only bacteria with surface-displayed biotin are selected.
Once a clear enrichment of bacteria is observed, the isolated clones can be further mutated and thereby develop highly active BirA variants that allow for specific biotinylation of native proteins.
