Plant Pectates Lyase Recombinant Expression In Escherichia Coli

Our lab has successfully expressed the polygalacturonase which from Aspergillus. oryzae and the pectate lyases which from Aspergillus. niger by using the Escherichia coli protein expression system. We found that they can act on the petin which from plant materials and promote plant cell walls to expansion. So we try to find out if the same kind of enzymes that from plants has the same function or even better. Since the expression of pectate lyases is difficult, which result in the limitation of their researches and applications . We found an impotant pectate lyase P56 coming from tomato by searching NCBI database with the conserved sequences of Pel A (coming from Aspergillus. nidulans) being successfully expressed in Escherichia coli. Their conserved sequences have 90% identity. P56 was the first pectate lyase found in plant kingdom by Wing etal in 1989, no paper reports the expression of this protein in any organisms.We try to use the Escherichia coli protein expression system to express the pectate lyase P56 in order to identify its activity. The whole cDNA of P56 was aquired from tomato genomic DNA by over-lap PCR methord because it was hard to aquire the material. The cDNA was inserted into three different vectors (PET 28a(+), PET 32a(+) and PET 43.1b(+), respectively), constructing recombinant plasmid (PET 28a(+)/L56, PET32a(+)/L56, PET43.1b(+)/L56), then transformed into host cell Escherichia coli. BL21-CodenPlus(DE3)-RIL. Protein was expressed under the induce of 0.5 mM IPTG for 48 h, at 15℃ and 180 rpm. The cells were sonicated on ice and pellected. Then the supernatant was purified with Ni-NTA His · Bind Resins and dialyzed against 10 mM sodium acetate, pH 7.5 with dialysis membrane. P56 protein was cut from the fused protein Nus-P56 by digesting with enterokinase. P56 is expected to act on polygalactuoronic acid and produce product with reducing polysaccharide at one end and unsaturated bond at another end. We did not find any enzyme activity in all last...