Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors
© Kang et al; licensee BioMed Central Ltd. 2008
Received: 20 June 2008
Accepted: 11 December 2008
Published: 11 December 2008
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.
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/K M ) of the precursor proteins by two orders of magnitude.
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.
Multicellular organisms maintain homeostasis through a balance between cell proliferation and death. Apoptosis is a controlled cell death process crucial in a wide range of biological activities, such as normal cell turnover, immune system, embryonic development, metamorphosis, and chemical-dependent cell death . Neuronal death due to aberrant apoptosis underlies the symptoms of various neurological disorders, such as Alzheimer's, Parkinson's and Huntington's diseases, stroke, amyotropic lateral sclerosis (ALS), multiple sclerosis (MS) and spinal muscular atrophy . On the other hand, inactivation of apoptosis by blocking upstream death signals or inhibition of caspase activity by IAP complex formation is central to cancer development and cellular resistance of cells against anticancer agents [3–5].
Caspases, a family of cysteine proteases, play crucial roles in apoptosis, pro-inflammatory cytokine activation, and presumably, keratinocyte differentiation [6, 7]. Following the initial identification of caspase-1 in 1992 by two different groups [8, 9], eleven caspases in humans and 25 in other eukaryotes have been characterized over the last decade . In mammals, caspases are translated as inactive zymogens. While caspases-8 and -10 are activated by death receptor-mediated signals (extrinsic apoptosis pathway), caspase-9 activity is stimulated by intracellular death signals, including cytochrome c released from mitochondria (intrinsic apoptosis pathway). Activated caspases subsequently convert procaspase-3 and -7 to fully active enzymes by specific proteolytic cleavage. Caspase-6 is activated after caspase-3. The three former caspases are known as apoptotic initiators, whereas the latter three are known as apoptotic effectors or executioners. Caspase-3 (also designated CED-3, murine ICE, and a protease resembling ICE/CPP32 in humans) is the first reported apoptotic effector, and cleaves the majority of cellular substrates in apoptotic cells [11–14]. Caspase-7 is very similar to caspase-3 in terms of structure and substrate specificity . As caspase-3 and -7 are the final executioners of apoptosis, both inhibition and activation of catalytic activities are of significant interest as therapeutic strategies for neurodegenerative diseases and cancers [5, 15–18].
Drug discovery research, including screening chemical libraries as well as structural and kinetic analyses, requires large-scale caspase-3 preparation. When expressed in E. coli, full-length caspase-3 (procaspase-3) undergoes presumable autoprocessing to yield the appropriate subunits characteristic of the active enzyme with only a marginal expression level, probably due to its cytotoxicity . While full-length caspase-3 has been expressed in Pichia pastoris , the process is long and the yield is inadequate, compared to conventional protein expression method in E. coli. The most frequently employed large-scale caspase-3 preparation method includes separate expression of the two insoluble domains in E. coli and subsequent refolding of the two combined domains for the active enzyme . This method showed significantly improved protein yield. However, such a costly and time-consuming refolding process is unsuitable for efficient large-scale production for drug discovery research. Here we describe a novel method for high-level expression and purification of caspase-3 precursors. The precursor was strategically engineered to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3. Furthermore, this protein efficiently digested endogenous caspase-3 substrate.
Results and discussion
Design of thrombin-activatable caspase-3 precursors for high-level expression in E. coli
Expression and purification of the engineered caspase-3 precursors
Activation of engineered caspase-3 precursors
Kinetic analysis of caspase-3 precursors and activated proteins
Catalytic parameters of the caspase-3 mutants
14.16 ± 1.5
7.21 ± 0.93
(5.0 ± 0.1) × 105
16.38 ± 3.6
3.39 ± 0.58
(2.1 ± 0.1) × 105
93.20 ± 33.2
0.05 ± 0.02
(5.0 ± 0.0) × 102
93.49 ± 9.2
0.02 ± 0.00
(1.7 ± 0.0) × 102
18.09 ± 1.3
1.26 ± 0.03
(6.7 ± 0.9) × 104
Δ28/175TS processed with thrombin
41.13 ± 1.9
1.86 ± 0.09
(4.5 ± 0.0) × 104
28TS/175TS processed with thrombin
27.81 ± 5.7
0.78 ± 0.06
(2.9 ± 0.4) × 104
28TS/180TI processed with thrombin
21.39 ± 1.9
1.77 ± 0.13
(8.3 ± 0.1) × 104
Significant changes in kcat, rather than K M values induced by mutation or activation resulted in similar substrate binding in all protein mutants, but altered catalytic activity. The K M and kcat values of protein III were 93.2 μM and 0.05 s-1, while those of VI were 41.3 μM and 1.86 s-1, respectively. The data correspond to 2.3 and 37.2-fold changes in substrate binding affinity and catalytic activity, respectively, upon activation. These findings imply that mutation or activation processes do not significantly alter the global shape of the active site, but affect the spatial arrangement of catalytic Cys163, in agreement with a previous report showing that activation of caspase-7 leads to rearrangement of a loop containing the catalytic cysteine .
In this study, we demonstrate a novel strategy for a large-scale preparation of active caspase-3 in E. coli. Thrombin-activatable caspase-3 precursors were designed to suppress autoprocessing, and their activation regulated by thrombin. The designed precursors were highly expressed in E. coli, easily purified, and activated by thrombin digestion, yielding 10–15 mg of active caspase-3 from 1 L of E. coli culture. Following thrombin activation, catalytic activity was increased about 100-fold, analogous to previous findings on the wild-type precursor and mature caspase-3 protein. This research represents the first example to highly express a full-length caspase-3 as a soluble protein in E. coli, facilitating a large-scale preparation of active caspase-3. This system may be effectively applied to prepare other caspases on a large scale.
PCR primers used for plasmid construction
5' → 3'
Generation of caspase-3-overexpressing constructs
Wild-type caspase-3 gene was amplified using the forward primer, 5'-GGGAATTCCATATGGAGAACACTGAAAACTCAG-3' (P1) containing an NdeI site (marked in bold type letters), and reverse primer, 5'-CCGCTCGAGGTGATAAAAATAGAGTTCTTTTGT-3' (P2), with a XhoI site (underlined), and inserted into the corresponding sites of the pET21a plasmid (Novagen). The resulting construct was designated plasmid I. A Δ28 caspase-3 deletion mutant devoid of the N-terminal 28 amino acids of wild-type caspase-3 (I) was prepared using the forward primer, 5'-GGGAATTCCATATGTCTGGAATATCCCTGGACAACAGT-3' (P3) with an NdeI site (marked in bold type letters), and reverse primer, P2. The resulting DNA was inserted into the pET21a expression vector, and denoted plasmid II. For preparation of Δ28/175TS caspase-3, the 175TS mutant of plasmid I was initially prepared using the megaprimer PCR method with slight modifications . N-terminal megaprimer DNA encoding amino acids 1~177, which is substituted at positions 172~177 (IETDSG) with LVPRGS, was prepared using the forward primer, P1, and reverse primer, 5'-ATCAACGCTGCCGCGCGGCACCAGGCCACAGTCCAGTTCTGTACCACG-3' (P4). C-terminal megaprimer DNA substituted at positions 172–177 (IETDSG) with LVPRGS was prepared using the forward primer, 5'-TGTGGCCTGGTGCCGCGCGGCAGCGTTGATGATGACATGGCGTGTCAT-3', (P5) and reverse primer, P2. The thrombin recognition sites in P4 and P5 are italicized. Each megaprimer was extended by PCR, using the other as a template. The resulting DNA was inserted into pET21a using the NdeI and XhoI restriction sites, and designated plasmid IX. The Δ28/175TS caspase-3 mutant of plasmid IX was prepared using forward P3 and reverse P2 primers. The amplified product was inserted into the NdeI and XhoI sites of pET21a, and the construct denoted plasmid III. For generating the 28TS/175TS mutant caspase-3 construct in plasmid IX, the megaprimer with substitutions at positions 24–30 (LVPRGS in place of ESMDSG) was prepared using the forward primer, P1, and reverse primer, 5'-ACTGTTGTCCAGGGATATGCTGCCGCGCGGCACCAGGCTTCCATGTATGATCTT-3' (P6). The thrombin recognition site in P6 is italicized. The resulting megaprimer, in combination with P2, was applied for the next PCR reaction to prepare 28TS/175TS caspase-3 DNA, which was cloned into pET21a, and designated plasmid IV. The 28TS/D175A/180TI caspase-3 construct was generated in plasmid I using a combination of megaprimer PCR and the QuikChange site-directed mutagenesis kit (Stratagene). To prepare the 28TS caspase-3 construct in plasmid I, a corresponding megaprimer produced using the P1 forward and P6 reverse primers was employed together with P2. The resulting construct was designated plasmid X. A 28TS/D175A caspase-3 construct in plasmid X was prepared with the QuikChange kit using the forward primer, 5'-GACTGTGGCATTGAGACAGCGAGTGGTGTTGATGAT-3' (P7), and reverse primer, 5'-CAGATCATCAACACCACTCGCTGTCTCAATGCCACA-3' (P8), and designated plasmid XI. The Ala mutation sites in P7 and P8 are italicized. The thrombin recognition site was successfully inserted into the plasmid XI background with the megaprimer method. N- and C-terminal megaprimers were prepared using the forward primer, P1, and reverse primer, 5'-TATTTTATGACACGCCATGTCGCTGCCGCGCGGCACCAGATCATCAACACCACT-3' (P9), as well as the forward primer, 5'-ACAGACAGTGGTGTTGATGATCTGGTGCCGCGCGGCAGCGACATGGCGTGTCATAAA-3' (P10) and reverse primer, P2, respectively. The thrombin recognition sites are italicized. Each prepared megaprimer was extended by PCR, using the other as a template, and the resulting product inserted into the NdeI and XhoI sites of pET21a (designated plasmid V).
Preparation of recombinant caspase-3 precursors
E. coli BL21 Rosetta cells (Novagen) containing the specified expression plasmids were grown at 37°C in LB medium until an A600 of 0.6–0.8. Engineered caspase-3 precursors were expressed by adding 1 mM IPTG at 18°C for 18 h. Cells were harvested, washed with buffer A (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, and 0.05% β-mercaptoethanol), and lysed by ultrasonication. After centrifugation (29,820 g for 30 min), the supernatant was incubated with a cobalt affinity resin (TALON®, Clontech) on a rocker at 4°C for 1 h, and washed with buffer A containing 10 mM imidazole. Proteins were eluted from the metal affinity resin with buffer A supplemented with 100 mM imidazole. Following dialysis against 20 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, and 1 mM dithiothreitol, caspase-3 precursors were concentrated to 2 mg/ml and stored at -80°C.
Activation of caspase-3 precursors by thrombin
Caspase-3 precursors were activated by digestion with bovine thrombin. Briefly, 100 μg of each precursor protein was incubated with 1 NIH unit of thrombin in 50 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol, 3 mM CaCl2 at 4°C. Digestion was performed for 18 h, and monitored by 15% SDS-PAGE.
Treatment of caspase-3 precursors with wild-type caspase-3
Caspase-3 precursors were treated with wild-type caspase-3 to test autocleavage during activation. Briefly, 100 μg of each precursor protein was incubated with 1 μg of wild-type caspase-3 in 50 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol, and 5 mM DTT at RT. Cleavage was performed for 18 h, and monitored by 15% SDS-PAGE.
Determination of caspase-3 activity and kinetic constants
The activity of each caspase-3 protein was measured using the colorimetric substrate, Ac-DEVD-pNA. Briefly, caspase-3 was activated in reaction buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, and 5 mM dithiothreitol) for 18 h before use. The appropriate amount of activated caspase-3 (final concentrations of 10–80 nM) was added to the substrate solution at a series of final concentrations (0, 12.5, 25, 50, 100 and 200 μM). The p-nitroanilide released by the caspase reaction was monitored at 405 nm using a DU 800 UV-VIS Spectrophotometer (Beckman Coulter). Km and Vmax values were obtained using Hyper32 version 1.0.0 http://homepage.ntlworld.com/john.easterby/hyper32.html, and kcat values calculated from Vmax and the used enzyme concentration.
Cleavage of endogenous PARP by engineered caspases
Human promyelocytic leukemia HL60 cells were cultured in RPMI. If necessary, apoptosis was induced by treatment of the cells with 100 μM etoposide for 12 h. Cell lysates (60 μg) were incubated with engineered caspases at 37°C for 2 h. Then, the cleavage of endogenous PARP was determined by immunoblot analysis with an anti-PARP antibody. For negative control, cell lysates treated together with engineered caspases and z-DEVD-fmk were used [26, 27].
This work was supported by the R&D Program for Fusion Strategy of Advanced Technologies (MKE), the Bio-signal Analysis Technology Innovation Program (M10645010003-06N4501-00310) of the Ministry of Education, Science and Technology (MEST), and the Korea Science and Engineering Foundation (KOSEF).
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