Authors

A Guevara¹; C Lugo²; A Montilla²; M Calabokis²; J C Martinez²; J Ferreira²; J Bubis²;  ¹ Universidad Simon Bolivar, UK;  ² Universidad Simón Bolívar, Venezuela

Discussion

The full gene sequence encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum was cloned, and a poly-His tagged construct was also generated. Both, the wid type and poly-His tagged recombinant proteins were expressed in Escherichia Coli. Most of the recombinant PKAC1 protein was highly insoluble and was probably packaged into inclusion bodies. When bacterial homogenates were separated into its soluble and particulate fractions, a polypeptide of about 38-40 KDa was predominantly seen in the insoluble fraction, whereas a band of approximately 45-50 KDa was primarily contained in the soluble fraction. In an attempt to solubilize the recombinant parasite protein, the non-ionic, non-denaturing surfactants, N-octyl-B-D-glucopyranoside (OG, 19 nM) and N-octanoyl-N-methylglucamine (Mega 8, 79 nM), were initially incorporated into the lysis buffer, either alone or in combination; however, these detergents were not appropiate for solubilizing the PKAC1 protein. We also tried the anionic detergent N-laurylsarcosine (aka sarkosyl), and the treatment with 3% sarkosyl enhanced the solubilization of the expressed trypanosome protein although some insoluble material still remained. Incorporation of the non-ionic surfactant Triton X-100 (4%) into the sarkosyl containing lysis buffer yielded a slight increase in the solubilization of the protein. On the basis of these findings, we used a mixture of 3% sarkosyl, 4% triton X-100 and Mg-ATP (15 nM, MgCl₂ and 5 nM ATP) to solubilize the PKAC1 protein. We observed that the addition of MgATP during bacterial homogenization improved the solubilization of the recombinant PKAC1. Moreover, the soluble PKAC1 mainly corresponded to the form of the protein that migrated as a 45-50 KDa polypeptide, indicating that this higher band was probably produced by covalent phosphorylation. The kinase enzymatic activity of the expressed protein was measured with an electrophoretic gel-shift assay that employs a fluorescently modified synthetic heptapeptide known as kemptide (sequence: LRRASLG), which is a specific substrate for PKA and PKA-like enzymes. The recombinant protein was expressed in a functional form given that it was capable of phosphorylating kemptide. The poly-His tagged PKAC1 was purified by affinity chromatography using a Ni²⁺-chelating resin. In addition, a recombinant purification strategy based on the formation of hybrid  holoenzymes between  the recombinant T. equiperdum wid type PKAC1 and mammalian PKA regulatory subunits was employed for the purification of the parasite PKAC1 protein. By immobilizing the hybrid holoenzyme complexes on a Ni²⁺-chelatingaffinity resin, the T. equiperdum PKAC1 protein was specifically purified using high concentrations of cAMP.