Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

Abstract

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 lmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30°C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae

“…Recombinant protein expression in Escherichia coli provides the opportunity to get ample amount of recombinant proteins swiftly [ 15 ]. Nowadays, pET23a (+) is a widely used vector for the expression and purification of recombinant proteins [ 16 ]. Although the expression and production of recombinant proteins are a well-established phenomenon, certain obstacles can affect the process [ 17 , 18 ].…”