Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Abstract

Background: Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful.

Results: Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10-15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM) of the precursor proteins by two orders of magnitude.

Conclusion: A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

Figure 1

High-level expression of caspase-3 precursors in E. coli. A. Design of thrombin-activatable caspase-3 precursors resistant to autoactivation. B. Structure of designed proteins. I: wild-type caspase-3; II: caspase-3 devoid of the N-terminal 28 amino acids; III: Six amino acids (172–177) of construct II were substituted with LVPRGS (a sequence susceptible to thrombin activity); IV: Two sites (amino acid sequences 25–30 and 172–177) of wild-type caspase-3 were replaced with LVPRGS. V: Six amino acids (172–177) of wild-type caspase-3 were mutated to LVPRGS, and an additional LVPRGS motif inserted between Asp180 and Asp181. Asp175 was mutated to Ala.

Figure 2

SDS-PAGE analysis of engineered caspase-3 proteins. A. Expression and Western blot analysis of engineered caspase-3 proteins. Cell lysates (20 μg) were prepared using an identical method, and loaded onto each well. M, molecular weight size marker; lane 1, uninduced E. coli lysate expressing wild-type caspase-3; lane 2, IPTG-induced E. coli lysate expressing wild-type caspase-3 (I); lane 3, IPTG-induced E. coli lysate expressing Δ28-caspase-3 (II); lane 4, IPTG-induced E. coli lysate expressing Δ28/175TS-caspase-3 (III); lane 5, IPTG-induced E. coli lysate expressing 28TS/175TS-caspase-3 (IV); lane 6, IPTG-induced E. coli lysate expressing 28TS/180TI-caspase-3 (V). The right and left images were obtained after coomassie blue staining and Western blot analysis, respectively. B. Treatment of caspase-3 precursors with wild-type caspase-3 (I). After each caspase-3 precursor was treated with wild-type caspase-3 (I) at RT for 18 h, 20 μl of each sample was heat-denatured and analyzed by SDS-PAGE. M, molecular weight size marker; lane 1, III; lane 2, III processed with I; lane 3, IV; lane 4, IV processed with I; lane 5, V; lane 6, V processed with I; lane 7, C163S caspase-3; lane 8, C163S caspase-3 treated with I. C. Activation of caspase-3 precursors by thrombin. Caspase-3 precursors were treated with thrombin at 4°C for 18 h, and 20 μl of each sample was heat-denatured and analyzed using PAGE. M, molecular weight size marker; lane 1, III; lane 2, III processed with thrombin (VI); lane 3, IV; lane 4, IV processed with thrombin (VII); lane 5, V; lane 6, V processed with thrombin (VIII); lane 7, C163S caspase-3; lane 8, C163S caspase-3 processed with thrombin.

Figure 3

Comparison of engineered caspases with endogenous caspase-3 and cleavage of PARP by engineered caspases. A. Western blot analysis using anti-caspase-3 antibody. H, unstimulated HL-60 cell lysate; E, HL-60 cell lysate stimulated with etoposide; lane 1, purified wild-type caspase-3 (I); lane 2, purified II; lane 3, purified III; lane 4, VI; lane 5, purified IV; lane 6, VII; lane 7, purified V; lane 8, VIII. B. Western blot analysis of PARP cleavage by wild-type and engineered caspases (VI and VIII). HL-60 cell lysates were treated with each caspase in the presence and absence of caspase-3 inhibitor (z-DEVD-fmk) and Western blot analysis was carried out using an anti-PARP antibody which recognizes the full length (115 kDa) PARP and its cleavage product (24 kDa). H, unstimulated HL-60 cell lysate; E, HL-60 cell lysate stimulated with etoposide.