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Use of Pseudomonas aeruginosa Chaperones in Folding an Insect Recombinant Prolyl Endoprotease (rPEP) Expressed in Escherichia coli.
Author(s) -
Gautam Ranjeeta,
Dareddy Vanitha,
Clack Beatrice
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.784.7
Subject(s) - escherichia coli , chaperone (clinical) , plasmid , pseudomonas aeruginosa , enzyme , western blot , complementary dna , recombinant dna , biochemistry , microbiology and biotechnology , protease , biology , chemistry , bacteria , dna , gene , genetics , medicine , pathology
Chaperone assist in protein folding. The purpose of this study were to 1) determine if co‐expression of Pseudomonas aeruginosa GroESL or Trigger factor with prolyl endo protease (PEP) cDNA in Escherichia coli system resulted in increase amount of soluble active enzyme 2) determine if two different temperatures, 37°C for 24 hours and biphasic temperature incubation of 37°C for 6 hours followed by overnight in 28°C would increase in amount of soluble active PEP and 3) to identify which chaperone mechanism results in highest expression of active protein. GroESL and TF chaperones were cloned into pLys‐S and pLys‐E plasmid. The resulting pGROELS‐S/E plasmid or pTF‐S/E plasmid was co‐transformed into E. coli . Western Blot techniques were used to determine the level of protein expression. Enzyme activity was assayed using a synthetic chromagenic peptide. Biphasic temperature growth resulted in higher level of active than constant 37°C. pGroESL‐ E and pTF‐S resulted in highest increase in amount of soluble rPEP as compared to amount found. Expression in presence of pGroESL‐E and pTF‐S resulted in the highest levels of active soluble enzyme. P. aeruginosa GroESL and TF chaperones co‐express and assist in folding successfully expression of a large insect PEP in E. coli . Funding: Office of Research and Sponsored Projects, Stephen F Austin State University; Ed and Gwen Cole, Nacogdoches, TX.