Mutazyme 2

The M13 filamentous phage display vector, fUSE55, was a gift from George P. Smith, University of Missouri, Columbia, MO (GenBank Accession AF317217). fUSE55 phage with in frame cDNA insertions express a fusion protein as part of the wild-type PIII protein with a valency of 3–5 copies per phage particle. A library of mutant VWF73 sequences was constructed for expression in the fUSE55 phage display system. In PCR reactions with VWF cDNA,[20 (link)] primer P1 and P2, Table 1 amplified the D1596 to R1688 region of VWF and added NH₂-terminal FLAG and T7 epitope tags. Mutagenic PCR was performed according to previously published methods [21 (link)]. Alternatively, to create a third library, mutagenic PCR was performed using the same primers and template but using the Mutazyme II (Agilent, Santa Clara, CA) kit, according to the manufacturer’s instructions. The mutated VWF73 PCR products were cloned into the BglI restriction site of fUSE55. Ligation products were electroporated into MC1061 cells [22 (link)]. The library depth was quantified by the number of colony forming units per mL (CFU) on NZY plates supplemented with 40 μg/mL tetracycline. Colonies were randomly selected to confirm VWF73 insertion by DNA sequencing using primer P3 and P4, Table 1.

Mutagenic PCR employing Mutazyme II (Agilent) was performed to amplify proA genes from the pTrcHisB into which they had been cloned. The purified PCR fragments were digested with NheI and BamHI and religated into pTrcHisB (Life Technologies). The plasmid was then introduced into electrocompetent 10β cells (New England Biolabs), and the cells plated on LB agar containing 50 µg/ml ampicillin. The cells were grown overnight at 37°C. The following morning, about 10⁵ colonies were suspended in 1 ml of LB and plasmid DNA was extracted using a Qiagen plasmid mini kit.
The resulting libraries were introduced into the ΔargC ΔproA strain by electroporation. Transformants were plated on M9/glucose containing proline (1 mM) and incubated overnight at 37°C. The next day, the largest colonies were picked from each plate and plasmid DNA was isolated. The inserted proA genes were sequenced by Macrogen.