Figure 2
From: A high-throughput, restriction-free cloning and screening strategy based on ccd B-gene replacement
ccd B-gene replacement is an efficient cloning strategy in obtaining active, recombinant OXA-protein. A) Lane 1 shows the PCR products from bla OXA-181 gene amplification analysed by agarose gel electrophoresis. M, Perfect DNA 1 KB ladder; relevant bands are indicated to the right. Arrow indicates the product matching the expected size, 857 bp. B) Lane 1 shows the PCR products from the bla OXA-181 gene inserted into pDEST17 by exponential plasmid amplification and analysed on agarose gel. M, Perfect DNA 1 KB ladder; relevant bands are indicated to the right. Arrow indicates the plasmid product matching the expected size, 5408 bp. C) Lane 1–14 shows the results from colony PCR analysis of clones 1–14. The expected size of the screened inserts is 948 bp, and arrow indicates the position of products matching the expected size. D) SDS-PAGE analysis of the expression of recombinant OXA-48. Lane 1 shows the uninduced control, whereas lane 2 shows the IPTG-induced recombinant OXA-48 protein expressed in E. coli strain BL21Star(DE3)pRARE. M, Precision Plus protein standard; relevant bands are indicated to the right. Arrow indicates the position of induced protein matching the theoretical mass of OXA-48 with the leader sequence removed (28 kDa). E) Recombinant OXA-48 protein was isolated from the periplasm, and purified through two anionic exchange steps. The integrity of the purified protein is shown in lane 1. M, Precision Plus protein standard; relevant bands are indicated to the right. F) The β-lactam antibiotic, meropenem, is hydrolysed by OXA-48. The hydrolysis velocities (μM/s) were plotted as a function of Meropenem concentration (μM); the enzyme follows the Michaelis-Menten kinetics. Based on these data, K m was calculated to be 18 ± 5 μM and k cat to 0.23 ± 0.02 s−1, giving a catalytic efficiency of 1.2 × 104 (M−1 s−1).