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Expression of highly active proteins using a cell-free human IVT system
Active caspase-3 produced by cell-free protein expression with the 1-Step Human High-Yield In Vitro Translation Kit is more functional than recombinant caspase-3 expressed in bacteria.
Krishna Vattem, Ph.D.;
In vitro translation (IVT) systems have been used for rapid expression of functional proteins, including enzymes. Typically, proteins expressed in mammalian cells have higher activity compared to those expressed in bacteria, because slower translational rates in the mammalian systems indirectly influence better folding of proteins. The Thermo Scientific 1-Step Human High-Yield IVT System is based on HeLa cell extracts that contain the necessary mammalian cellular machinery for protein expression and folding. Here, we demonstrate the ability of this IVT system to rapidly express highly functional caspase-3 enzyme, as well as how it can be used to screen modulators of protein activity.
Caspase-3 protein belongs to the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. Caspase-3, in particular, (also known as CPP32/Yama/apopain) is formed from a 32kDa zymogen that is cleaved into 17kDa and 12kDa subunits [1-3]. Our results demonstrate a 3-fold higher activity for caspase-3 expressed in human IVT compared to a recombinant protein expressed and purified from bacteria.
RESULTS and DISCUSSION:
We cloned the caspase-3 gene into pT7CFE1-CHis expression vector (Part No. 88860) and then expressed the protein using the 1-Step Human High-Yield Mini IVT Kit (Part No. 88891). To assess the resulting expression and activity levels of caspase-3, we performed a Western blot (Figure 1) and an activity assay (Figure 2) to compare the IVT product to known amounts of a commercially available purified recombinant caspase-3 from E. coli. Overexpression of caspase-3 is known to induce proteolytic self-cleavage into 17kDa and 12kDa subunits, and it is the 17kDa fragment that is indicative of active protein and which is detected by our anti-caspase-3 antibody . Thus, the Western blot (Figure 1) demonstrates a high level of expression of active (cleaved) caspase-3 by the Human IVT Kit.
Figure 1. Western blot demonstrates high-yield IVT expression of Caspase-3. Western blot of caspase-3 in samples electrophoresed as purified recombinant protein (Sigma-Aldrich)(left) and as reaction-aliquots from the 1-Step Human High-Yield Mini IVT Kit (Part No. 88891)(right).
Figure 2. Caspase-3 expressed using the human IVT system is more active than a purified protein expressed in bacteria. Equal amounts of human caspase-3 purified from E. coli or expressed with the 1-Step Human High-Yield IVT Kit were assayed for active caspase-3 activity using Caspase-glo™ Assay Reagent mix containing cleavable DEVD-aminoluciferin and luciferase substrate, according to instructions of the manufacturer (Promega Corp.).
The activity of the IVT-expressed caspase-3 was measured using an assay mix containing cleavable DEVD-aminoluciferin and luciferase substrate and was found to be 3-fold higher compared to E. coli expressed and purified caspase-3 (Figure 2). Ac-DEVD-CHO is a synthetic tetrapeptide inhibitor for caspase-3 and contains the amino acid sequence of the PARP cleavage site. The tetrapeptide inhibitor can be used to identify and quantify the caspase-3 activity in apoptotic cells, and to study events downstream of caspase-3 activation [5-7].
We have demonstrated that human IVT expressed caspase-3 was completely inhibited by 1µM of a Ac-DEVD-CHO, compared to no inhibition obtained when using a negative control Z-FA-FMK inhibitor (Figures 3 and 4). These results suggest that one can not only rapidly express highly active caspase-3 using human IVT system but also utilize this system to carry out small molecule inhibitor screens to identify caspase-3 modulators.
Figure 3. Specific inhibition of cell free expressed caspase-3 using Ac-DEVD-CHO inhibitor. Aliquots (0.1µL) of caspase-3 expressed with the 1-Step Human High-Yield IVT Kit were incubated with 1µM of Ac-DEVD-CHO (inhibitor), 1µM Z-FA-FMK (off-target inhibitor) or no chemical (none) for 10 minutes and then measured for caspase-3 activity using the Caspase-glo™ Assay Reagent (Promega Corp.).
Figure 4. Determination of IC50 value for caspase specific inhibitor Ac-DEVD-CHO. Various concentrations of Ac-DEVD-CHO were added to 1µL of high-yield IVT reactions expressing caspase-3 in a total volume of 100µL. Relative light units (RLU) values were plotted against concentrations of AC-DEVD-CHO to obtain IC50 values.
We have successfully demonstrated that Human IVT can be used to express active caspase-3 protein that can be inhibited by known synthetic tetrapeptides such as Ac-DEVD-CHO making this system amenable to screening for caspase-3 modulators.
Expression of caspase-3
Reactions (25µL) containing 50% human cell lysate, 2.5µL Accessory Proteins, 5µL Reaction Mix, and 1µg caspase-3 DNA cloned into pCFE-CHis expression vector were incubated at 30°C for 6 hours according to instructions provided in the kit (Part No. 88891).
At the end of incubation, 1, 2 and 4µL of samples were boiled in SDS-PAGE sample buffer and run on a 4-12 % SDS-PAGE gel. In parallel, 1, 2 and 4µg of recombinant caspase-3 (#C1224, Sigma-Aldrich Co.) were also run in the same SDS-PAGE gel. Western immunoblots were carried out using anti-caspase-3 antibody and the Fast Western Blot SuperSignal West Dura Mouse Kit (Part No. 35070).
For measuring the activity of caspase-3 enzyme, equal amounts of either human caspase-3 purified from E. coli or expressed using human high-yield IVT systems were assayed using Caspase-glo™ Assay Reagent mix containing cleavable DEVD-aminoluciferin and luciferase substrate according to the manufacturer instructions (Promega Corp.). For determining the specificity, 0.1µL of human high-yield IVT-expressed caspase-3 was incubated with 1µM of caspase-3 specific inhibitor Ac-DEVD-CHO (#235420, EMD Millipore Corp.), off-target inhibitor Z-FA-FMK (#342000, EMD Millipore Corp.), or no chemical (none) for 10 minutes and measured for caspase-3 activity using Caspase-glo™ Assay Reagent. For determining IC50 values of caspase-3 expressed in human IVT, various concentrations of Ac-DEVD-CHO were added to 1µL of high-yield reactions expressing caspase-3 in a total volume of 100µL. Relative light units (RLU) values were plotted against concentration of AC-DEVD-CHO to obtain IC50 values.
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