Different Fusion Expression Of Full-length PDE4B2 And Truncat For Characterization

Phosphodiesterase(PDE)catalyzes hydrolysis of cAMP and cGMP as intracellular second messengers.The human PDE4 isoenzymes selective hydrolyze cAMP and have four subtypes of PDE4 A,4B,4C and 4D with characteristics of different tissue-specific distribution.The mononuclear and neutrophils involved in the inflammatory response mainly express PDE4B2.Therefore,PDE4 inhibitors can be used to treat asthma,chronic obstructive pulmonary disease and other inflammatory diseases.A mixture-based screen of ligand liberary using the magetic-bund target has been developed,which needed a large quantity of activity protein.The soluble expression PDE4B2 in prokaryotic cells was low yield and was unstable during purification,which can not meet the requirement of PDE4B2 for screening of potential antiinflammatory activity inhibitors.At the same time,full-length human PDE4B2 was found existing in two binding conformations against R-rolipram: the high affinity binding conformation against R-rolipram related to the toxic side effects of PDE4B2 inhibitors while the low affinity binding conformation against R-rolipram related to the therapeutic effect.Therefore,to provide plenty of fusion PDE4B2 bearing high activity and low affinity binding conformation against R-rolipram for high throughput screening of PDE4 inhibitor with potential antiinflammatory activity,different tagged fusion expressions of full-length human PDE4B2 and its 152-528 aa truncates were compared.Firstly,the full length of human hPDE4B2 and the 152-528 aa truncate were fused with 6His-SUMO and expressed in Escherichia coli and purified by Ni2+-NTA to obtain the full-length SF-hPDE4B2 and truncated mutant ST-hPDE4B2.Western blot was used to detect the peptide composition.The activity of the enzyme was determined by calf intestinal alkaline phosphatase(CIAP)coupled malachite green(MLG)phosphorus method,and further characteraed by the Km and inhibitory constants for the model compounds.The results indicated the apparent specific activity of ST-hPDE4B2 was nearly 40-fold higher than that of SF-h PDE4B2,and the activity yield of ST-hPDE4B2 was over 100-fold higher than of SF-hPDE4B2.The total yield of this expression was not high,and Western blot showed that there were many degradation fragments.Inhibition constant of(R)-Rolipram indicated that SF-hPDE4B2 mainly existed in the high-affinity state while ST-hPDE4B2 appeared principally in the low-affinity state for(R)-Rolipram.By the comparision analyses of inhibition constants of some represntaive compounds against the two target enzymes,a monotonical correlation by a double inverse model was found.Therefore,compared with SF-hPDE4B2,ST-hPDE4B2 may be more advantags for the initial screening of anti-inflammatory activity hPDE4B2 inhibitors.Secondly,the hPDE4B2 full length and 152-528 aa truncated mutant gene were cloned into GST-tagged vector pGEX-6P-1 by homologous recombination in Escherichia coli and purified by GST affinity chromatography to obtain the N-terminal GST-tagged full-length hPDE4B2(GF-hPDE4B2)and truncate(GT-hPDE4B2)However,the maximum apparent specific activity of GF-hPDE4B2 was 0.01 U·mg-1,and the maximum specific activity of GF-hPDE4B2 was 0.05 U·mg-1.GF-hPDE4B2 and GT-hPDE4B2 have low activity and are not suitable for high throughput screening of PDE4 inhibitors.In order to increase the soluble expression abundance of hPDE4B2,and explore the ditag purification method.MBP and 6His were used on N-terminal and/or C-terminal fusion expression of hPDE4B2 as N-MBP/6His-C,N-6His/MBP-C and N-6H-MBP/6His-C.Then full length hPDE4B2 and 152-528 aa truncates were cloned into the corresponding vector plasmids by homologous recombination,and MCF-hPDE4B2 / MCT-hPDE4B2,MNF-hPDE4B2 / MNT-hPDE4B2 MF-hPDE4B2 / MT-hPDE4B2 plasmids were obtain.After expression,the lysates of enzymes were purified by affinity chromatography through Ni2+-NTA and MBP.The apparent specific activities were as follows: MCF-hPDE4B2(1.11 U·mg-1)> MF-hPDE4B2(0.1 U·mg-1)>MNF-hPDE4B2(0.01 U·mg-1),and MCT-hPDE4B2(1.25 U·mg-1)>MNF-hPDE4B2(0.6 U·mg-1)>MF-hPDE4B2(0.3 U·mg-1);PDE4B2 full length and truncated mutant recombinant protein also had yieldes significantly higher than the other two fusion approaches.Using MCF-hPDE4B2 and MCT-hPDE4B2 as the target protein will have significant advantage of cost.In summary,full length hPDE4B2 and truncates fused with three tags in different patterms were obtained,and were characterized for the enzymatic features and inhibition constants of the typical inhibitors.The results showed that the active yield of ST-hPDE4B2 was better than that of SF-hPDE4B2;those of MCF-hPDE4B2 and MCT-hPDE4B2 were the best.Additionally,full length hPDE4B2 was predominantly in high affinity conformation for binding(R)-Rolipram,while hPDE4B2 truncate was predominantly in low affinity binding conformation.Therefore,MCT-hPDE4B2 can be used for high-throughput screening of potential anti-inflammatory activity PDE4 inhibitors,and MCF-hPDE4B2 for their verification.