The authors would like to acknowledge Dr. Evette Radisky and Alexandra Hockla at the Mayo Clinic in Jacksonville, Florida, for providing the pET-3a-pro-MMP-3cd plasmid as the template for cloning the Hisx6pro-MMP-3cd gene, and their comments, along with Dr. Paul Hartley from the Nevada Genomics Center at the University of Nevada, Reno, for DNA sequencing. The authors would also like to thank Cassandra Hergenrader for helping with part of protein expression. M.R.-S. would like to thank the NIH-P20 GM103650-COBRE Integrative Neuroscience grant and the UNR R&#38;D mICRO SEED Grant Award.

1. MMP expression
<ol>
	<li>Cloning and transformation of pET-3a-Hisx6-pro-MMP-3cd into R2DP cells
	<ol>
		<li>Digest the pET-3a plasmid (see the Table of Materials) with NdeI and BamHI restriction enzymes in Digest Buffer (see the Table of Materials). In a total reaction volume of 40 &#181;L, add 4 &#181;L of Digest Buffer, 33 &#181;L of 100 ng/&#181;L plasmid, and 1.5 &#181;L of each restriction enzyme and allow the reaction to proceed for ~2 h until completion at 37 &#176;C.</li>
		<li>Perform a PCR reaction on the MMP-3cd sequence to insert an N-terminal His-tag. Use 25 &#181;L of PCR Mix (see the Table of Materials), 2.5 &#181;L of 10 &#181;M primers (Supplemental Figure S1), and 1.25 &#181;L of the 100 ng/&#181;L insert sequence. Add sterile water to a final reaction volume of 50 &#181;L.</li>
		<li>Run the PCR product and digested vector on a 1% agarose gel. Purify the gel bands using a Gel Recovery Kit (see the Table of Materials) per the manufacturer&#39;s protocol.</li>
		<li>Clone the amplified PCR product into the digested vector between the NdeI and BamHI restriction sites using DNA Assembly Mix (see the Table of Materials). Use online tools to determine the required volume of the insert and cut vector for a total reaction volume of 15 &#181;L.</li>
		<li>Thaw a 50 &#181;L aliquot of high-transformation efficiency cells (see the Table of Materials) on ice until thawed. Prewarm SOC Growth Medium (see the Table of Materials) to 37 &#176;C and LB-ampicillin (LB Amp) plates (see the Table of Materials).</li>
		<li>Add 1-2 &#181;L of the pET-3a-Hisx6-pro-MMP-3cd assembly reaction to the 50 &#181;L aliquot. Incubate on ice for 30 min.</li>
		<li>Heat-shock the cells by incubating at 42 &#176;C for 30 s. Incubate on ice for 2 min.</li>
		<li>Add 950 &#181;L of SOC growth medium to each transformant mixture. Shake for 1 h at 250 rpm and 37 &#176;C.</li>
		<li>Plate 100 &#181;L of the transformants on LB Amp plates and incubate overnight at 37 &#176;C.</li>
		<li>Inoculate each isolated colony in 10 mL of LB Amp medium. Shake overnight at 250 rpm and 37 &#176;C.</li>
		<li>Extract plasmid DNA per manufacturer&#39;s protocol for the miniprep kit (see the Table of Materials). Confirm the sequence of the construct using T7 forward and reverse primers (Supplemental Figure S1). 
		NOTE: The pET-3a-Hisx6-pro-MMP-3cd construct DNA can be stored at -20 &#176;C. When ready, proceed with transformation into R2DP cells.</li>
		<li>Thaw one 20 &#181;L aliquot of R2DP cells (see the Table of Materials) on ice for 2-5 min. Prewarm SOC growth medium to room temperature and LB Amp CamR plates to 37 &#176;C (see the Table of Materials).</li>
		<li>Add 1 &#181;L of 100 ng/&#181;L sequence-confirmed pET-3a-Hisx6-pro-MMP-3cd to the 50 &#181;L aliquot. Stir gently to mix and return the tube to ice.</li>
		<li>Incubate the tube on ice for 5 min.</li>
		<li>Heat-shock the cells by incubating at 42 &#176;C for exactly 30 s. Do not shake.</li>
		<li>Place the cells on ice for 2 min.</li>
		<li>Add 80 &#181;L of room-temperature SOC medium to the transformant mixture. Shake for 1 h at 250 rpm and 37 &#176;C.</li>
		<li>Plate the transformants on LB Amp CamR plates and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Growth and induction
	<ol>
		<li>Inoculate a single, isolated colony of R2DP pET-3a-Hisx6-pro-MMP-3cd transformant from an LB Amp CamR plate in 10 mL of LB Amp CamR media at 37 &#176;C. Shake at 250 rpm overnight (~16 h). Save aliquots from each culture and prepare 40% (v/v) glycerol (see the Table of Materials) stocks if desired.</li>
		<li>Per overnight culture, inoculate a 1 L flask containing 500 mL of LB Amp CamR medium to an optical density at 600 nm (OD600) of 0.05-0.1. 
		NOTE: This should return the cells to logarithmic growth.</li>
		<li>Measure the OD600 at several time points, typically for 3-4 h, until it falls between 0.4 and 0.6.</li>
		<li>Before induction, aliquot a fraction of culture into a 1.5 mL microfuge tube (see the Table of Materials) and label it Un-induced Fraction. Store it at -80 &#176;C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.5.</li>
		<li>Induce the cultures to a final concentration of 1 mM using 1 M isopropyl-&#223;-D-thiogalactopyranoside (IPTG) stock (see the Table of Materials). Continue to incubate in the 37 &#176;C shaker for an additional 3-4 h. 
		NOTE: During expression, the reader should determine the optimal OD600 at the time of induction and the IPTG concentration. If the yield drops substantially after purification, the imidazole concentration in purification buffers may require adjustment, or the cell pellet may need to be sonicated further.</li>
		<li>Before centrifuging the cultures, aliquot a fraction of culture into a second 1.5 mL microfuge tube and label it Induced Fraction. Store it at -80 &#176;C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.7.</li>
		<li>Centrifuge the cell culture in 250 mL conical bottles (see the Table of Materials) at maximum speed and 4 &#176;C for 10 min.</li>
		<li>Repeat step 1.2.7 until the cultures are completely pelleted. 
		NOTE: PAUSE: Cell pellets can be frozen at -80 &#176;C and thawed later for further processing. Otherwise, skip this step and proceed to step 1.3.1.</li>
	</ol>
	</li>
	<li>Inclusion body extraction and solubilization 
	NOTE: Prepare fresh 10 M urea, preferably no earlier than one day in advance, stirring thoroughly until dissolved completely. Do not heat or autoclave urea; store it at room temperature.
	<ol>
		<li>Resuspend the pellet (from step 1.2.8) in lysis buffer (see the Table of Materials). Per gram of pellet, add 3 mL of lysis buffer and resuspend by vortexing or pipetting. Shake overnight at 4 &#176;C.</li>
		<li>Add 1.25 mL of 10% (w/v) sodium deoxycholate (see the Table of Materials) per 1 L of culture. Shake at room temperature for 30 min at 150 rpm.</li>
		<li>Add 10 &#181;L of DNase I (see the Table of Materials) per 1 L of culture. Shake at room temperature for 30 min at 150 rpm.</li>
		<li>Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Set aside a fraction of Lysed MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.6. 
		NOTE: After centrifugation, the pellet may be stringy and not compactly packed, making it risky to discard the supernatant. If this is the case, then skip step 1.3.6 and proceed to step 1.3.7.</li>
		<li>Discard the supernatant from the centrifuged samples. 
		NOTE: The protocol can be paused at this point and the cell pellets frozen at -80 &#176;C and thawed later. Otherwise, skip this step and proceed to step 1.3.7.</li>
		<li>Resuspend the pellet in 100 mL/L culture of Inclusion Body Buffer (see the Table of Materials) by pipetting up and down.</li>
		<li>During sonication, keep the samples on ice to prevent overheating. Sonicate each sample for 6 cycles of 15 s, output 5, and 50% pulse. Allow 15 s rest periods for cooling between cycles. 
		NOTE: If necessary, transfer the samples into 50 mL conical tubes for further centrifugation (see the Table of Materials). Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Set aside a fraction of Sonicated MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.11.</li>
		<li>Check the pellet. If stringy, repeat steps 1.3.8-1.3.10. If the pellet is compact, discard the supernatant and proceed to step 1.3.12. 
		NOTE: Sonication in Inclusion Body Buffer can be repeated to recover more protein from the lysed cell debris. However, too much sonication can cause shearing, which harms MMP yield. The protocol can be paused at this stage, and the cell pellets can be frozen at -80 &#176;C and thawed later.</li>
		<li>Resuspend each pellet from a 1 L culture in 5 mL of Solubilization Buffer (see the Table of Materials) by pipetting. Incubate for at least 30 min on ice to allow the proteins to solubilize.</li>
		<li>Set aside a fraction of Solubilized MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.14.</li>
		<li>Centrifuge the cells for 10 min at 13,000 &#215; g and 4 &#176;C. DO NOT DISCARD THE SUPERNATANT.</li>
		<li>If a pellet forms/remains after centrifugation, pour the supernatant into a separate 50 mL conical tube. Resuspend the pellet in another 5 mL of Solubilization Buffer (per 1 L of culture) by pipetting up and down.</li>
		<li>Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C. DO NOT DISCARD THE SUPERNATANT.</li>
		<li>Repeat steps 1.3.13 and 1.3.14 until a little to no pellet forms after centrifugation or only gray precipitate remains. Pool the supernatants. Discard or store the pellet at -80 &#176;C for additional sonication.</li>
	</ol>
	</li>
</ol>
2. MMP purification and refolding
<ol>
	<li>His-tag (HT) affinity purification
	<ol>
		<li>Per the manufacturer&#39;s protocol, fill a gravity-flow column (see the Table of Materials) with well-mixed Ni-NTA resin (see the Table of Materials). Allow the resin to settle and separate from the storage buffer such that a distinct line forms between the two layers. 
		NOTE: Never allow the resin to dry, as air will penetrate the resin and harm protein yield. In between uses, perform the resin regeneration procedure described in section 2.2.</li>
		<li>Allow the storage buffer to drain. Fill the column with two resin-bed volumes of HT Equilibration Buffer.</li>
		<li>Drain the HT Equilibration Buffer and discard. As the column is draining, centrifuge the protein extract at 13,000 &#215; g for 1 min and filter-sterilize using a 0.22 &#181;m filter (see the Table of Materials).</li>
		<li>Swap the waste container for a 50 mL conical tube labeled HT Flowthrough. Add the prepared protein extract to the column.</li>
		<li>Reapply the flowthrough to maximize binding.</li>
		<li>Set aside a fraction of Flowthrough Fraction for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.7.</li>
		<li>Immediately wash the resin with 15 mL of HT Wash Buffer (see the Table of Materials). Collect the flowthrough in 15 mL conical tubes (see the Table of Materials) labeled HT Wash. 
		NOTE: Absorbance values at 280 nm (A280) were obtained via spectrophotometry and used along with molecular weight and extinction coefficient, &#949;, to estimate protein concentrations. For denatured Hisx6-pro-MMP-3cd, the molecular weight is 29.86 kDa, and &#949; is 34.38 M-1 cm-1.</li>
		<li>Blanking against HT Wash Buffer, measure and record the A280. Repeat steps 2.1.7 and 2.1.8 with additional wash fractions. Once the A280 approaches baseline and impurities have been minimized, proceed to step 2.1.9.</li>
		<li>Set aside a fraction of Wash Fraction for gel analysis. Repeat for multiple wash fractions. Store the fractions at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.10.</li>
		<li>Immediately elute His-tagged proteins by adding 5 mL of HT Elution Buffer (see the Table of Materials). Collect the flowthrough as 0.5-1 mL fractions in microfuge tubes labeled HT Elution.</li>
		<li>Set aside a fraction of Elution Fraction for gel analysis. Repeat for multiple elution fractions. Store the fractions at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.12.</li>
		<li>If the A280 is &#62;0.3 mg/mL, dilute the fraction with HT Equilibration Buffer (see the Table of Materials). 
		NOTE: The eluted fraction must be diluted to an A280 of 0.3 mg/mL or less to prevent precipitation during dialysis. The protocol can be paused here and the pooled fractions frozen at -80 &#176;C and thawed later. Otherwise, skip this step and proceed to step 2.2.1.</li>
	</ol>
	</li>
	<li>Resin regeneration
	<ol>
		<li>Wash the resin with ten resin-bed volumes of HT Regeneration Buffer (see the Table of Materials) and ten resin-bed volumes of sterile water.</li>
		<li>Store the resin as a 50% slurry in 20% (v/v) ethanol in water.</li>
	</ol>
	</li>
</ol>
3. Protein refolding
NOTE: For smaller volumes, dialysis cassettes can be used at a lower risk of sample loss. Dialysis tubing is required if larger volumes are used (see the Table of Materials).
<ol>
	<li>Dialysis 
	NOTE: The optimum protein concentration for dialysis is ~0.3 mg/mL. If significant precipitation occurs during dialysis, reduce the urea concentration gradient between each dialysis using a stepwise dialysis method and add more intermediate steps (e.g., from 6 M to 5 M, and then 5 M to 4 M, rather than skipping the 5 M stage). As freeze-thaw cycles damage the cellular and protein structure, it is vital to minimize pauses in the protocol.
	<ol>
		<li>Per the manufacturer&#39;s protocol, use the appropriate amount of dialysis tubing according to the volume of Elution Fraction samples.</li>
		<li>Submerge the eluted MMP fractions in dialysis tubing in 1 L of Dialysis Buffer 1 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer to 1 L of Dialysis Buffer 2 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer to 1 L of Dialysis Buffer 3 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer the sample into new 50 mL conical tubes and label them as Dialyzed MMP.</li>
		<li>Examine the tube for any precipitate. If precipitate has formed, centrifuge the sample for 1 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Transfer the supernatant into new 15 mL conical tubes and label them as Refolded MMP.</li>
		<li>Set aside a fraction for gel analysis and label it as Refolded MMP. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 3.1.9. 
		NOTE: If yields are low, the precipitate can be dissolved in HT Equilibration Buffer and steps in section 3.1 repeated with dialysis tubing. If gel analysis is not to be performed or if the protocol must be paused here, freeze the samples at -80 &#176;C and thaw them later. If yields are in the desired range, proceed to step 3.2.1.</li>
	</ol>
	</li>
	<li>Reconcentration 
	NOTE: The extinction coefficients for refolded and denatured Hisx6-pro-MMP-3cd are expected to be the same; hence, A280 calculations are not affected.
	<ol>
		<li>Reconcentrate the sample up to 0.5 mg/mL. Use a 400 mL stirred cell (see the Table of Materials) to concentrate the sample to 15 mL. To prevent foaming, use a 50 mL reconcentration tube to concentrate further if needed. 
		NOTE: If a precipitate forms, it can be pelleted and dissolved in HT Equilibration Buffer. Then, repeat sections 3.1 and step 3.2.1. Otherwise, continue to step 3.2.2.</li>
		<li>Set aside a fraction for gel analysis and label it as Concentrated MMP. 
		&#8203;NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later.</li>
	</ol>
	</li>
</ol>
4. Activation
<ol>
	<li>4-Aminophenylmercuric acetate (APMA) activation 
	NOTE: APMA is highly toxic. Make a fresh stock solution of 20 mM APMA before activation, and always work under a fume hood when using APMA. Discard the APMA waste into its container.
	<ol>
		<li>Per 1 mL aliquot of MMP (1 mg/mL), add 50 &#181;L of 20 mM APMA (see the Table of Materials) to reach a final APMA concentration of 1 mM. Incubate overnight at 37 &#176;C.</li>
		<li>If a precipitate forms, centrifuge it at maximum speed for 10 min at 4 &#176;C. Store the supernatant in a 1.5 mL microfuge tube labeled Activated MMP. Discard the precipitate into a container marked for APMA waste.</li>
		<li>Set aside a fraction for gel analysis and label it Activated MMP. 
		NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later. If not performing gel analysis, proceed to step 4.2.1. After activation, the molecular weight and extinction coefficient of MMP-3cd are 19.40 kDa and 28.42 M-1 cm-1, respectively.</li>
	</ol>
	</li>
	<li>Desalting
	<ol>
		<li>Remove APMA from the activated MMP-3cd sample with a 2 mL desalting column (see the Table of Materials), following the manufacturer&#39;s protocol.</li>
		<li>Set aside a fraction for gel analysis and label it Desalted MMP. Store it at -80 &#176;C. If not performing gel analysis, proceed to section 4.3 with the remaining samples. 
		NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later.</li>
	</ol>
	</li>
	<li>Running the SDS-PAGE gels
	<ol>
		<li>Run all protein fractions on SDS-PAGE gels: Un-induced Fraction, Induced Fraction, Lysed MMP, Sonicated MMP, Solubilized MMP, Flowthrough Fraction, Wash Fraction, Elution Fraction, Refolded MMP, Concentrated MMP, Activated MMP, and Desalted MMP.</li>
	</ol>
	</li>
	<li>Long-term storage of MMP-3
	<ol>
		<li>Add 0.05% (v/v) nonionic surfactant (see the Table of Materials) to the desalted MMP-3cd samples and store them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Portolano, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+for+structural+biology+in+HEK+293F+suspension+cells:+A+novel+and+accessible+approach.">Recombinant protein expression for structural biology in HEK 293F suspension cells: A novel and accessible approach.</a> Journal of Visualized Experiments: JoVE. (92), e51897 (2014).</li><li>Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. W., Barb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+yield+expression+of+recombinant+human+proteins+with+the+transient+transfection+of+HEK293+cells+in+suspension.">High yield expression of recombinant human proteins with the transient transfection of HEK293 cells in suspension.</a> Journal of Visualized Experiments: JoVE. (106), e53568 (2015).</li><li>Nilvebrant, J., Alm, T., Hober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal+protein+purification+facilitated+by+a+small+bispecific+affinity+tag.">Orthogonal protein purification facilitated by a small bispecific affinity tag.</a> Journal of Visualized Experiments: JoVE. (59), e3370 (2012).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+production+of+soluble+proteins+from+insoluble+inclusion+bodies+by+a+two-step-denaturing+and+refolding+method.">Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.</a> PLoS One. 6 (7), 22981 (2011).</li><li>Hu, X., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+functional+matrix+metalloproteinases+by+zymography.">Detection of functional matrix metalloproteinases by zymography.</a> Journal of Visualized Experiments: JoVE. (45), e2445 (2010).</li><li>Radisky, E. S., Raeeszadeh-Sarmazdeh, M., Radisky, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+matrix+metalloproteinase+inhibition+in+breast+cancer.">Therapeutic potential of matrix metalloproteinase inhibition in breast cancer.</a> Journal of Cellular Biochemistry. 118 (11), 3531-3548 (2017).</li><li>Raeeszadeh-Sarmazdeh, M., Do, L. D., Hritz, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors:+Potential+for+the+development+of+new+therapeutics.">Metalloproteinases and their inhibitors: Potential for the development of new therapeutics.</a> Cells. 9 (5), 1313 (2020).</li><li>Nagase, H., Visse, R., Murphy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+matrix+metalloproteinases+and+TIMPs.">Structure and function of matrix metalloproteinases and TIMPs.</a> Cardiovascular Research. 69 (3), 562-573 (2006).</li><li>Raeeszadeh-Sarmazdeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+the+metalloproteinase+inhibitor+TIMP-1+reveals+that+its+N-+and+C-terminal+domains+cooperate+in+matrix+metalloproteinase+recognition.">Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition.</a> Journal of Biological Chemistry. 294 (24), 9476-9488 (2019).</li><li>Batra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-10+(MMP-10)+interaction+with+tissue+inhibitors+of+metalloproteinases+TIMP-1+and+TIMP-2.">Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2.</a> Journal of Biological Chemistry. 287 (19), 15935-15946 (2012).</li><li>Singh, K. K., Jain, R., Ramanan, H., Saini, D. K., Galea, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+matrix+metalloproteinases+in+Escherichia+coli.">Expression and purification of matrix metalloproteinases in Escherichia coli.</a> Matrix Metalloproteases. , 3-16 (2017).</li><li>Manka, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+triple-helical+collagen+cleavage+by+matrix+metalloproteinase+1.">Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (31), 12461-12466 (2012).</li><li>Gomis-Ruth, F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+inhibition+of+the+human+matrix+metalloproteinase+stromelysin-1+by+TIMP-1.">Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.</a> Nature. 389 (6646), 77-81 (1997).</li><li>Shirian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converting+a+broad+matrix+metalloproteinase+family+inhibitor+into+a+specific+inhibitor+of+MMP-9+and+MMP-14.">Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14.</a> FEBS Letters. 592 (7), 1122-1134 (2018).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+histidine-tagged+catalytic+domain+of+MMP-13+in+one+step+using+affinity+column+and+renaturation+of+it+with+histidine+tag.">Purification of recombinant histidine-tagged catalytic domain of MMP-13 in one step using affinity column and renaturation of it with histidine tag.</a> Journal of Liquid Chromatography &amp; Related Technologies. 37 (15), 2118-2130 (2014).</li><li>Aydin, H., Azimi, F. C., Cook, J. D., Lee, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+and+general+expression+platform+for+the+production+of+secreted+proteins+from+human+cells.">A convenient and general expression platform for the production of secreted proteins from human cells.</a> Journal of Visualized Experiments: JoVE. (65), e4041 (2012).</li><li>McNiff, M. L., Haynes, E. P., Dixit, N., Gao, F. P., Laurence, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioredoxin+fusion+construct+enables+high-yield+production+of+soluble,+active+matrix+metalloproteinase-8+(MMP-8)+in+Escherichia+coli.">Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.</a> Protein Expression and Purification. 122, 64-71 (2016).</li><li>Maity, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GST-His+purification:+A+two-step+affinity+purification+protocol+yielding+full-length+purified+proteins.">GST-His purification: A two-step affinity purification protocol yielding full-length purified proteins.</a> Journal of Visualized Experiments: JoVE. (80), e50320 (2013).</li><li>Stefan, A., Ceccarelli, A., Conte, E., Mont&#243;n Silva, A., Hochkoeppler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+benefits+of+protein+co-expression+in+Escherichia+coli.">The multifaceted benefits of protein co-expression in Escherichia coli.</a> Journal of Visualized Experiments: JoVE. (96), e52431 (2015).</li><li>Yadavalli, R., Sam-Yellowe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HeLa+based+cell+free+expression+systems+for+expression+of+Plasmodium+rhoptry+proteins.">HeLa based cell free expression systems for expression of Plasmodium rhoptry proteins.</a> Journal of Visualized Experiments: JoVE. (100), e52772 (2015).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+magnetosome+associated+protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 magnetosome associated protein MamA&#916;41.</a> Journal of Visualized Experiments: JoVE. (37), e1844 (2010).</li></ol>

The authors declare that they have no competing financial interests.

The large-scale production of soluble, human, recombinant MMPs remains a challenging task. Mammalian cells can express functional MMPs at high costs and long wait times, whereas E. coli rapidly produce high quantities of MMP inclusion bodies that must be purified and refolded11,16. R2DP cells significantly increase the yield of MMP inclusion bodies, enabling a more cost-effective and productive MMP refolding process. However, E. coli lack the po...

Most complex eukaryotic proteins undergo elaborate posttranslational modifications after expression, requiring highly assisted protein folding and co-factors to be functional1. Producing large amounts of soluble human protein in a bacterial host remains a significant challenge due to high costs and the lack of robust expression and purification methods, even for smaller-scale laboratory experiments2,3. MMPs, human endopeptidases with large molecular weight, are usually expressed as insoluble inclusion bodies when expressed in E. coli. Extraction of soluble human MMPs often leads to a laborious, time-consuming solubilization and refolding process4.
MMPs have critical roles in both physiological and pathogenic processes. Human MMPs are a family of 23 zinc endopeptidases, categorized by structure and substrate specificity, and differentially expressed in spite of a highly conserved catalytic domain5,6. MMPs are secreted as inactive zymogens, regulated via posttranslational activation and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs)7,8,9,10. Though initially recognized for their role in ECM turnover, MMPs have also been implicated in development, morphogenesis, tissue repair, and remodeling8. Dysregulation of MMPs has been notably linked to cancer along with neurodegenerative, cardiovascular, and fibrotic diseases, among other illnesses5,7.
The development of robust large-scale MMP production methods is critical to ensure the success of future studies of MMP mechanisms through biochemical and cell-based assays. Various MMPs have been previously expressed in bacteria11, including Hisx6-tagged MMPs, without altering MMP activity12,13,14,15. However, these methods include tedious, long steps that might be difficult to replicate.
Mammalian cells can also be used to express many different human proteins while ensuring the proper posttranslational modifications16. Although the mammalian expression system is an ideal choice to produce recombinant human proteins with proper post-translational modifications, the main disadvantages of this method are initial low yields, costly growth media and reagents, long timelines to reach stable expression lines, and risk of contamination with other species such as fungi or bacteria2,11. Moreover, MMP production in mammalian cell lines yields impurities from associated cellular proteins such as TIMPs or fibronectins11. Unlike the slow cell growth observed in mammalian cells, the bacterial expression system offers large-scale protein production in a short period along with simpler media and growth requirements. However, due to the lack of other associated cellular proteins (i.e., TIMPs) in bacterial expression systems, active MMPs at higher concentrations are subject to degradation through autoproteolysis, resulting in poor MMP yield17.
This paper describes a detailed method for bacterial expression, purification, and activation of recombinant Hisx6-pro-MMP-3cd using E. coli as an expression host due to its affordability, simplicity, and success in producing higher yields of MMPs2,3,18. Since E. coli lacks the protein folding machinery and posttranslational processing required for recombinant MMPs and other complex proteins, many E. coli strains have been engineered to overcome these limitations, making E. coli a more suitable host for expression of recombinant human MMP-3cd,19,20. For instance, the R2DP strain used in this study enhances eukaryotic expression by supplying a chloramphenicol-resistant plasmid containing codons rarely used in E. coli.
As described in this protocol, after overexpression of relatively pure inclusion bodies from the pET-3a vector (Figure 1) in R2DP cells, Hisx6-pro-MMP-3 catalytic domain (MMP-3cd) proteins are extracted and denatured4. Hisx6-pro-MMP-3cd3,19 was purified using&#160;affinity tag chromatography. Upon refolding and dialysis, the pro-MMP-3cd (zymogen) was activated by 4-aminophenylmercuric acetate (APMA), and SDS-PAGE analysis is used to evaluate yields and the need for further purification5,21. This protocol describes expression, purification, and activation of soluble MMP-3cd as an example. However, it may be also used as a guide for expression of other MMPs and human proteases with similar expression, and activation mechanisms (Figure 2). For other proteins other than MMP-3cd, the reader is advised to determine optimal buffer compositions and methods for their target protein before attempting this protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63263/63263fig01.jpg" /> 
Figure 1: Plasmid map of the pET-3a-Hisx6-pro-MMP-3cd plasmid. The pET-3a vector includes an ampicillin resistance gene. An N-terminal Hisx6-tag sequence is cloned into the pET-3a-based vector, including pro-MMP-3cd, to yield the pET-3a-Hisx6-pro-MMP-3cd construct under control of T7 promoter between BamHI and NdeI restriction sites. <a href="https://www.jove.com/files/ftp_upload/63263/63263fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63263/63263fig02.jpg" /> 
Figure 2: Bacterial expression of pro-MMP-3cd, purification, refolding, and activation. 1.1: pET-3a-Hisx6-pro-MMP-3cd plasmid was transformed into BL21(DE3) or R2DP Cells. 1.2: Pro-MMP-3cd protein expression was induced using IPTG. 1.3: Chemical lysis and sonication are used to extract Hisx6-pro-MMP-3cd proteins that are mainly insoluble and found in the inclusion bodies. Urea was used to denature and solubilize protein from inclusion bodies. 2.1. Denatured Hisx6-pro-MMP-3cd protein was purified via affinity chromatography purification. 3. The eluted Hisx6-pro-MMP-3cd was slowly refolded during dialysis through gradual removal of urea from the buffer. 4. Finally, refolded MMP-3cd protein was activated using APMA by removing the N-terminal pro-peptide domain. APMA is later removed from the solution through desalting. The numbers correspond to protocol sections describing these steps. Abbreviations: MMP-3cd = Matrix metalloproteinase-3 catalytic domain; APMA = 4-aminophenylmercuric acetate. <a href="https://www.jove.com/files/ftp_upload/63263/63263fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m sterile filter</td>
			<td>Sigma Aldrich</td>
			<td>SLGP033RS</td>
			<td>Used to remove some contaminants from the protein extract before purification, and prevent the Ni-NTA column from clogging</td>
		</tr>
		<tr>
			<td>1 L Erlenmeyer flasks</td>
			<td>Thermo Fisher Scientific</td>
			<td>S76106F</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>1 L glass bottles</td>
			<td>Thermo Fisher Scientific</td>
			<td>06-414-1D</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-682-002</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>18 G, 1-in. beveled needle</td>
			<td>Amazon</td>
			<td>B07S7VBHM2</td>
			<td>Used in combination with the dialysis casette</td>
		</tr>
		<tr>
			<td>2 mL desalting column</td>
			<td>Thermo Fisher Scientific</td>
			<td>89890</td>
			<td>Removes APMA following activation</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAA1610422</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>250 mL conical bottle cushions</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-538-53A</td>
			<td>Stabilize conical bottles during large-volume centrifugation</td>
		</tr>
		<tr>
			<td>250 mL conical bottles</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-538-53</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>400 mL stirred cell</td>
			<td>Sigma Aldrich</td>
			<td>UFSC40001</td>
			<td>Re-concentrates a much larger volume than the centrifugal filter unit. Rosetta2(DE3)pLysS cells produce high volumes of protein that may exceed the 15 mL limit of the centrifugal filter unit</td>
		</tr>
		<tr>
			<td>4-aminophenylmercuric acetate (APMA)</td>
			<td>Sigma Aldrich</td>
			<td>A9563-5G</td>
			<td>Activates MMP-3 by cleaving the propeptide</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0829167</td>
			<td>Used in combination with the dialysis casette</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td>Used for storage in many purification steps</td>
		</tr>
		<tr>
			<td>50 mL re-concentration tube</td>
			<td>Sigma Aldrich</td>
			<td>UFC901024D</td>
			<td>Used for re-concentrating protein samples after dialysis or removing contaminants</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1423-500</td>
			<td>Buffer ingredient that solidifies autoclaved LB media upon cooling</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1760-25</td>
			<td>Antibiotic used with pET3a vector; used at 100 &#181;g/mL in LB media</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>NEB</td>
			<td>R3136S</td>
			<td>Restriction enzyme to be used with the pET3a vector</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>600-30-23</td>
			<td>The calcium ion stabilizes MMP structure</td>
		</tr>
		<tr>
			<td>Cell spreaders</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-189-7544</td>
			<td>Can be used to spread cells across a petri dish after transformation</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-055-125GM</td>
			<td>Antibiotic used with pET3a vector; used at 34 &#181;g/mL in LB media</td>
		</tr>
		<tr>
			<td>Dialysis Buffer 1</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2, 1 &#181;M ZnCl2, 4 M Urea.</td>
		</tr>
		<tr>
			<td>Dialysis Buffer 2</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2, 1 &#181;M ZnCl2, 2 M Urea.</td>
		</tr>
		<tr>
			<td>Dialysis Buffer 3</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2 , 1 &#181;M ZnCl2.</td>
		</tr>
		<tr>
			<td>Dialysis clips</td>
			<td>Thermo Fisher Scientific</td>
			<td>68011</td>
			<td>Used in combination with snakeskin dialysis tubing</td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Thermo Fisher Scientific</td>
			<td>88243</td>
			<td>Alternative dialysis method that holds much larger sample volumes, but with higher risk of sample loss</td>
		</tr>
		<tr>
			<td>Digest buffer</td>
			<td>NEB</td>
			<td>B7204S</td>
			<td>Buffer used in digesting the pET3a vector</td>
		</tr>
		<tr>
			<td>Disposable cuvettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-200-257</td>
			<td>Used to measure the bacterial culture OD during growth and expression</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D107125G</td>
			<td>Assists with protein denaturation by reducing any disulfide bonds</td>
		</tr>
		<tr>
			<td>DNA assembly mix</td>
			<td>NEB</td>
			<td>E2621S</td>
			<td>Used to ligate the Hisx6-pro-MMP-3cd PCR product and digested pET3a vector</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>NEB</td>
			<td>M0303S</td>
			<td>Endonuclease for degrading unfavorable DNA contaminants that could later affect protein purification</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A995-4</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>J15694-AE</td>
			<td>Used in denaturation. Prevents oxidation and subsequent formation of disulfide bonds</td>
		</tr>
		<tr>
			<td>Gel recovery kit</td>
			<td>Promega</td>
			<td>A9281</td>
			<td>Isolates and purifies DNA from agarose gels</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>G33-500</td>
			<td>Used for making glycerol stocks, which are frozen at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Gravity flow column</td>
			<td>BioRad</td>
			<td>7321010</td>
			<td>Used for Ni-NTA purification of recombinantly His-tagged proteins</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride (GdnHCl)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAA135430B</td>
			<td>Second chaotropic agent used for disrupting protein secondary structure.</td>
		</tr>
		<tr>
			<td>High-transformation efficiency cells</td>
			<td>NEB</td>
			<td>C2987</td>
			<td>High-transformation efficiency cells with greater chance of success for cloning the N-terminal His-tag into the pET3a-pro-MMP-3cd construct</td>
		</tr>
		<tr>
			<td>HT Elution Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 250 mM imidazole. Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>HT Equilibration Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea. Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>HT Regeneration Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM MES, 0.1 M NaCl. Adjust pH to 5.0</td>
		</tr>
		<tr>
			<td>HT Wash Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 25 mM imidazole. Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A144C-212</td>
			<td>Used to pH buffers</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAA1022122</td>
			<td>Mimics the histidine side group. Used to separate non-specifically binding proteins from the his-tagged target protein</td>
		</tr>
		<tr>
			<td>Inclusion Body Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 5 mM DTT, 2% v/v Triton X 100, 0.5 M Urea. Adjust pH to 8.0</td>
		</tr>
		<tr>
			<td>Isopropyl-&#223;-D-thiogalactopyranoside (IPTG)</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERR0392</td>
			<td>A reagent that induces target gene expression in pET3a. Make 0.5 mL 1 M aliquots, filter sterilize and store in -20 &#176;C</td>
		</tr>
		<tr>
			<td>LB Amp CamR media</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>To be poured into a sterible 1 L bottle or 1 L flask. For 1 L, add 25 g LB Broth. Sterilize by autoclaving. Once cooled to below 50 &#176;C, add ampicillin to 100 &#181;g/mL and chloramphenicol to 34 &#181;g/mL</td>
		</tr>
		<tr>
			<td>LB Amp CamR plates</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>To be poured into sterile petri dishes. Pour until the petri dish lid is completely covered. 1 L of media yields 40-60 plates. For 1 L: 25 g LB Broth, 16 g Agar. Sterilize by autoclaving. Once cooled to below 50 &#176;C, add ampicillin to 100 &#181;g/mL and chloramphenicol to 34 &#181;g/mL</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1426-2</td>
			<td>Pre-mixed with tryptone, yeast extract, and sodium chloride</td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.133 g/mL lysozyme, 0.49% v/v Triton X-100. Adjust pH to 8.0</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>MP Biomedicals</td>
			<td>195303</td>
			<td>Used in protein extraction. Enzyme that lyses bacterial cell walls</td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Promega</td>
			<td>A1330</td>
			<td>If successful, extracts the pET3a-pro-MMP-3cd construct from transformants</td>
		</tr>
		<tr>
			<td>NdeI</td>
			<td>NEB</td>
			<td>R0111S</td>
			<td>Restriction enzyme to be used with the pET3a vector</td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>Thermo Fisher Scientific</td>
			<td>PI88221</td>
			<td>Used to bind recombinant his-tagged proteins. This strong interaction can be displaced with higher concentrations of imidazole</td>
		</tr>
		<tr>
			<td>PCR mix</td>
			<td>NEB</td>
			<td>M0492S</td>
			<td>A PCR reagent for inserting an N-terminal his-tag into the pET3a-pro-MMP-3cd vector</td>
		</tr>
		<tr>
			<td>pET plasmid</td>
			<td>Addgene</td>
			<td>n/a</td>
			<td>The pET3a vector offers ampicillin resistance, inducible expression of a target gene, and sequencing with T7 primers</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>Used for plating transformants on LB agar media</td>
		</tr>
		<tr>
			<td>R2DP cells</td>
			<td>Novagen</td>
			<td>714033</td>
			<td>BL21 derivatives with enhanced expression of eukaryotic proteins. Contain tRNAs of codons found to be rare in e. coli</td>
		</tr>
		<tr>
			<td>SOC growth media</td>
			<td>NEB</td>
			<td>B9020S</td>
			<td>Non-selective growth media for rapid growth during transformation</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP358-1</td>
			<td>Used in buffers and helps with protein stability</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Thermo Fisher Scientific</td>
			<td>PI89905</td>
			<td>Detergent used in protein extraction. Lyses cell walls</td>
		</tr>
		<tr>
			<td>Solubilization Buffer</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM DTT, 6 M Urea. Adjust pH to 8.0</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Common buffer used in the physiological pH range. Temperature-sensitive</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher Scientific</td>
			<td>M1122980101</td>
			<td>Detergent used for cell lysis</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ75826A7</td>
			<td>First chaotropic agent for disrupting protein secondary structure</td>
		</tr>
		<tr>
			<td>Zinc chloride (ZnCl2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAA162810E</td>
			<td>Stabilizes MMP structure. The zinc ion is found in the catalytic site of MMP-3</td>
		</tr>
	</tbody>
</table>

bacterial expression and purification of human matrix metalloproteinase-3 using affinity chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

When running samples on SDS-PAGE, because the protein is expressed in the form of insoluble inclusion bodies, the lysed and sonicated fractions should contain little to no Hisx6-pro-MMP-3cd extract, as the protein has not yet been resolubilized in urea. Figure 3 compares the His-tag purification elution fractions of Hisx6-pro-MMP-3cd from BL21(DE3) cells and R2DP cells. Elution fractions were pooled separately for both BL21(DE3) and R2DP cells before dialysis. Fractions from each step were r...

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Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

bacterial-expression-purification-human-matrix-metalloproteinase-3

Research

JoVE Journal

Biochemistry

4.4K Views.  University of Nevada, Reno. His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Video: Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.