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

摘要

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.

摘要

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.

引言

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 experiments^2,3. 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 domain^5,6. 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 illnesses^5,7.

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 activity^12,13,14,15. 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 bacteria^2,11. 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 MMPs^2,3,18. 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-3cd^3,19 was purified using 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 purification^5,21. 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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

方案

1. MMP expression

1. Cloning and transformation of pET-3a-Hisx6-pro-MMP-3cd into R2DP cells
   1. 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 µL, add 4 µL of Digest Buffer, 33 µL of 100 ng/µL plasmid, and 1.5 µL of each restriction enzyme and allow the reaction to proceed for ~2 h until completion at 37 °C.
   2. Perform a PCR reaction on the MMP-3cd sequence to insert an N-terminal His-tag. Use 25 µL of PCR Mix (see the Table of Materials), 2.5 µL of 10 µM primers (Supplemental Figure S1), and 1.25 µL of the 100 ng/µL insert sequence. Add sterile water to a final reaction volume of 50 µL.
   3. 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's protocol.
   4. 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 µL.
   5. Thaw a 50 µL 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 °C and LB-ampicillin (LB Amp) plates (see the Table of Materials).
   6. Add 1-2 µL of the pET-3a-Hisx6-pro-MMP-3cd assembly reaction to the 50 µL aliquot. Incubate on ice for 30 min.
   7. Heat-shock the cells by incubating at 42 °C for 30 s. Incubate on ice for 2 min.
   8. Add 950 µL of SOC growth medium to each transformant mixture. Shake for 1 h at 250 rpm and 37 °C.
   9. Plate 100 µL of the transformants on LB Amp plates and incubate overnight at 37 °C.
   10. Inoculate each isolated colony in 10 mL of LB Amp medium. Shake overnight at 250 rpm and 37 °C.
   11. Extract plasmid DNA per manufacturer'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 °C. When ready, proceed with transformation into R2DP cells.
   12. Thaw one 20 µL 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 Cam^R plates to 37 °C (see the Table of Materials).
   13. Add 1 µL of 100 ng/µL sequence-confirmed pET-3a-Hisx6-pro-MMP-3cd to the 50 µL aliquot. Stir gently to mix and return the tube to ice.
   14. Incubate the tube on ice for 5 min.
   15. Heat-shock the cells by incubating at 42 °C for exactly 30 s. Do not shake.
   16. Place the cells on ice for 2 min.
   17. Add 80 µL of room-temperature SOC medium to the transformant mixture. Shake for 1 h at 250 rpm and 37 °C.
   18. Plate the transformants on LB Amp Cam^R plates and incubate overnight at 37 °C.
2. Growth and induction
   1. Inoculate a single, isolated colony of R2DP pET-3a-Hisx6-pro-MMP-3cd transformant from an LB Amp Cam^R plate in 10 mL of LB Amp Cam^R media at 37 °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.
   2. Per overnight culture, inoculate a 1 L flask containing 500 mL of LB Amp Cam^R medium to an optical density at 600 nm (OD₆₀₀) of 0.05-0.1.
      NOTE: This should return the cells to logarithmic growth.
   3. Measure the OD₆₀₀ at several time points, typically for 3-4 h, until it falls between 0.4 and 0.6.
   4. 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 °C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.5.
   5. Induce the cultures to a final concentration of 1 mM using 1 M isopropyl-ß-D-thiogalactopyranoside (IPTG) stock (see the Table of Materials). Continue to incubate in the 37 °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.
   6. 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 °C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.7.
   7. Centrifuge the cell culture in 250 mL conical bottles (see the Table of Materials) at maximum speed and 4 °C for 10 min.
   8. Repeat step 1.2.7 until the cultures are completely pelleted.
      NOTE: PAUSE: Cell pellets can be frozen at -80 °C and thawed later for further processing. Otherwise, skip this step and proceed to step 1.3.1.
3. 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.
   1. 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 °C.
   2. 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.
   3. Add 10 µL of DNase I (see the Table of Materials) per 1 L of culture. Shake at room temperature for 30 min at 150 rpm.
   4. Centrifuge for 10 min at 13,000 × g and 4 °C.
   5. Set aside a fraction of Lysed MMP for gel analysis. Store it at -80 °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.
   6. Discard the supernatant from the centrifuged samples.
      NOTE: The protocol can be paused at this point and the cell pellets frozen at -80 °C and thawed later. Otherwise, skip this step and proceed to step 1.3.7.
   7. Resuspend the pellet in 100 mL/L culture of Inclusion Body Buffer (see the Table of Materials) by pipetting up and down.
   8. 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 × g and 4 °C.
   9. Set aside a fraction of Sonicated MMP for gel analysis. Store it at -80 °C. If not performing gel analysis, proceed to step 1.3.11.
   10. 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 °C and thawed later.
   11. 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.
   12. Set aside a fraction of Solubilized MMP for gel analysis. Store it at -80 °C. If not performing gel analysis, proceed to step 1.3.14.
   13. Centrifuge the cells for 10 min at 13,000 × g and 4 °C. DO NOT DISCARD THE SUPERNATANT.
   14. 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.
   15. Centrifuge for 10 min at 13,000 × g and 4 °C. DO NOT DISCARD THE SUPERNATANT.
   16. 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 °C for additional sonication.

2. MMP purification and refolding

1. His-tag (HT) affinity purification
   1. Per the manufacturer'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.
   2. Allow the storage buffer to drain. Fill the column with two resin-bed volumes of HT Equilibration Buffer.
   3. Drain the HT Equilibration Buffer and discard. As the column is draining, centrifuge the protein extract at 13,000 × g for 1 min and filter-sterilize using a 0.22 µm filter (see the Table of Materials).
   4. Swap the waste container for a 50 mL conical tube labeled HT Flowthrough. Add the prepared protein extract to the column.
   5. Reapply the flowthrough to maximize binding.
   6. Set aside a fraction of Flowthrough Fraction for gel analysis. Store it at -80 °C. If not performing gel analysis, proceed to step 2.1.7.
   7. 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, ε, to estimate protein concentrations. For denatured Hisx6-pro-MMP-3cd, the molecular weight is 29.86 kDa, and ε is 34.38 M^-1 cm^-1.
   8. 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.
   9. Set aside a fraction of Wash Fraction for gel analysis. Repeat for multiple wash fractions. Store the fractions at -80 °C. If not performing gel analysis, proceed to step 2.1.10.
   10. 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.
   11. Set aside a fraction of Elution Fraction for gel analysis. Repeat for multiple elution fractions. Store the fractions at -80 °C. If not performing gel analysis, proceed to step 2.1.12.
   12. If the A280 is >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 °C and thawed later. Otherwise, skip this step and proceed to step 2.2.1.
2. Resin regeneration
   1. 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.
   2. Store the resin as a 50% slurry in 20% (v/v) ethanol in water.

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).

1. 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.
   1. Per the manufacturer's protocol, use the appropriate amount of dialysis tubing according to the volume of Elution Fraction samples.
   2. 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 °C.
   3. 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 °C.
   4. 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 °C.
   5. Transfer the sample into new 50 mL conical tubes and label them as Dialyzed MMP.
   6. Examine the tube for any precipitate. If precipitate has formed, centrifuge the sample for 1 min at 13,000 × g and 4 °C.
   7. Transfer the supernatant into new 15 mL conical tubes and label them as Refolded MMP.
   8. Set aside a fraction for gel analysis and label it as Refolded MMP. Store it at -80 °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 °C and thaw them later. If yields are in the desired range, proceed to step 3.2.1.
2. 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.
   1. 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.
   2. Set aside a fraction for gel analysis and label it as Concentrated MMP.
      ​NOTE: The protocol can be paused here and the samples frozen at -80 °C and thawed later.

4. Activation

1. 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.
   1. Per 1 mL aliquot of MMP (1 mg/mL), add 50 µL of 20 mM APMA (see the Table of Materials) to reach a final APMA concentration of 1 mM. Incubate overnight at 37 °C.
   2. If a precipitate forms, centrifuge it at maximum speed for 10 min at 4 °C. Store the supernatant in a 1.5 mL microfuge tube labeled Activated MMP. Discard the precipitate into a container marked for APMA waste.
   3. 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 °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.
2. Desalting
   1. Remove APMA from the activated MMP-3cd sample with a 2 mL desalting column (see the Table of Materials), following the manufacturer's protocol.
   2. Set aside a fraction for gel analysis and label it Desalted MMP. Store it at -80 °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 °C and thawed later.
3. Running the SDS-PAGE gels
   1. 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.
4. Long-term storage of MMP-3
   1. Add 0.05% (v/v) nonionic surfactant (see the Table of Materials) to the desalted MMP-3cd samples and store them at -80 °C.

结果

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...

讨论

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 refolded^11,16. 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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
0.22 和微量;m 无菌过滤器	Sigma Aldrich	SLGP033RS	用于在纯化前去除蛋白质提取物中的一些污染物，并防止 Ni-NTA 柱堵塞
1 L 锥形瓶	Thermo Fisher Scientific	S76106F	n/a
1 L 玻璃瓶	Thermo Fisher Scientific	06-414-1D	n/a
1.5 mL 微量离心管	Thermo Fisher Scientific	02-682-002	不适用
15 mL 锥形管	Thermo Fisher Scientific	339650	n/a
18 G，1 英寸斜面针	Amazon	B07S7VBHM2	与透析盒结合使用
2 mL 脱盐柱	Thermo Fisher Scientific	89890	活化后去除 APMA
2-（N-吗啉代）乙磺酸 （MES）	Thermo Fisher Scientific	AAA1610422	n/a
250 mL 锥形瓶垫	Thermo Fisher Scientific	05-538-53A	在大容量离心过程中稳定锥形瓶
250 mL 锥形瓶	Thermo Fisher Scientific	05-538-53	n/a
400 mL 搅拌池	Sigma Aldrich	UFSC40001	再浓缩的体积比离心式过滤装置大得多。Rosetta2（DE3）pLysS 细胞产生大量蛋白质，可能超过离心过滤装置的 15 mL 限
4-氨基苯基乙酸汞 （APMA）	Sigma Aldrich	A9563-5G	通过切割前肽 5 mL 注射器激活 MMP-3
	Fisher Scientific	NC0829167	与透析盒
50 mL 锥形管	Thermo Fisher Scientific	339650	结合使用用于许多纯化步骤中的储存
50 mL 再浓缩管	Sigma Aldrich	UFC901024D	用于透析后重新浓缩蛋白质样品或去除污染物
琼脂	Thermo Fisher Scientific	BP1423-500	缓冲成分，可在冷却时固化高压灭菌的 LB 培养基
苄青霉素	Thermo Fisher Scientific	BP1760-25	与 pET3a 载体一起使用的抗生素;用于 100 µLB 培养基中的 g/mL
BamHI	NEB	R3136S	与 pET3a 载体一起使用的限制性内切酶
氯化钙 （CaCl2）	Thermo Fisher Scientific	600-30-23	钙离子稳定 MMP 结构
细胞铺展器	Thermo Fisher Scientific	50-189-7544	可用于在转化
后将细胞铺展到培养皿中氯霉素	Thermo Fisher Scientific	22-055-125GM	抗生素与 pET3a 载体一起使用;用于 34 µLB 培养基
透析缓冲液 1	n/a	n/a	20 mM Tris-HCl （pH 8.0）、150 mM NaCl、10 mM CaCl2、1 &微量;M ZnCl2，4 M 尿素。
透析缓冲液 2	n/a	n/a	20 mM Tris-HCl （pH 8.0）、150 mM NaCl、10 mM CaCl2、1 &微量;M ZnCl2，2 M 尿素。
透析缓冲液 3	n/a	n/a	20 mM Tris-HCl （pH 8.0）、150 mM NaCl、10 mM CaCl2、1 &微量;M ZnCl2 中。
透析夹	Thermo Fisher Scientific	68011	与蛇皮透析管结合使用
透析管	Thermo Fisher Scientific	88243	替代透析方法，可容纳更大的样品量，但样品损失风险更高
消化缓冲液	NEB	B7204S	用于消化 pET3a 载体的缓冲液
一次性比色皿	Thermo Fisher科学	21-200-257	用于测量细菌培养物生长和表达过程中的 OD
二硫苏糖醇 （DTT）Thermo	Fisher Scientific	D107125G	通过减少任何二硫键来帮助蛋白质变性
DNA 组装混合物	NEB	E2621S	用于连接 Hisx6-pro-MMP-3cd PCR 产物和消化的 pET3a 载体
DNase I	NEB	M0303S	核酸内切酶，用于降解以后可能影响蛋白质纯化的不利 DNA 污染物
乙醇	Thermo Fisher Scientific	A995-4	n/a
乙二胺四乙酸 （EDTA）	Thermo Fisher Scientific	J15694-AE	用于变性。防止氧化和随后形成二硫键
凝胶回收试剂盒	Promega	A9281	从琼脂糖凝胶中分离和纯化 DNA
甘油	Thermo Fisher Scientific	G33-500	用于制备甘油原液，将其在 -80 ° 下冷冻;C
重力流柱	BioRad	7321010	用于重组 His 标签蛋白的 Ni-NTA 纯化
高转化效率细胞	NEB	C2987	高转化效率细胞，将 N 端 His 标签克隆到 pET3a-pro-MMP-3cd 构建
HT 洗脱缓冲液	n/a	n/a	20 mM Tris-HCl （pH 8.0）、50 mM NaCl、6 M 尿素、250 mM 咪唑。将 pH 值调节至 7.4
HT 平衡缓冲液	n/a n	/a	20 mM Tris-HCl （pH 8.0）、50 mM NaCl、6 M 尿素。将 pH 值调节至 7.4
HT 再生缓冲液	n/a	n/a	20 mM MES，0.1 M NaCl。将 pH 值调节至 5.0
HT 洗涤缓冲液	n/a	n/a	20 mM Tris-HCl （pH 8.0）、50 mM NaCl、6 M 尿素、25 mM 咪唑。将 pH 值调节至 7.4
盐酸 （HCl）	Thermo Fisher Scientific	A144C-212	用于 pH 缓冲液
咪唑	Thermo Fisher Scientific	AAA1022122	模拟组氨酸侧基。用于将非特异性结合蛋白与 his 标记的靶蛋白
分离 包涵体缓冲液	n/a	n/a	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 尿素。将 pH 值调节至 8.0
异丙基-szlig;-D-硫代吡喃半乳糖苷 （IPTG）	Thermo Fisher Scientific	FERR0392	一种在 pET3a 中诱导靶基因表达的试剂。制备 0.5 mL 1 M 等分试样，过滤、消毒并储存在 -20 °C 中;C
LB Amp CamR media	n/a n	/a	倒入可消毒的 1 L 瓶子或 1 L 烧瓶中。对于 1 L，加入 25 g LB 肉汤。通过高压灭菌灭菌。冷却至 50 °C 以下;C、加入氨苄青霉素至100 &微量;g/mL 和氯霉素至 34 &微量;g/mL
LB Amp CamR 板	n/a	n/a	倒入无菌培养皿中。倒入直至培养皿盖完全覆盖。1 L 培养基可产生 40-60 个板。1 L 培养基：25 g LB 肉汤，16 g 琼脂。通过高压灭菌灭菌。冷却至 50 °C 以下;C、加入氨苄青霉素至100 &微量;g/mL 和氯霉素至 34 &微量;g/mL
LB 肉汤	Thermo Fisher Scientific	BP1426-2	与胰蛋白胨、酵母提取物和氯化钠
裂解缓冲液	n/a	n/a	预混 50 mM Tris-HCl （pH 8.0）、1 mM EDTA、100 mM NaCl、0.133 g/mL 溶菌酶、0.49% v/v Triton X-100。将 pH 值调节至 8.0
溶菌酶	MP 生物医学195303		用于蛋白质提取。裂解细菌细胞壁的酶
小量制备试剂盒	Promega	A1330	中提取 pET3a-pro-MMP-3cd 构建
			，与 pET3a 载体
Ni-NTA 树脂	Thermo Fisher Scientific	PI88221	一起使用用于结合重组 his 标签蛋白。这种强烈的相互作用可以用更高浓度的咪唑
非离子表面活性剂	Thermo Fisher Scientific	PI28316	Storage 去污剂来代替，以防止 MMP 聚集。最大限度地减少 MMP 表面疏水残基与水分子之间的相互作用，而不会破坏催化活性。
PCR 混合物	NEB	M0492S	一种 PCR 试剂，用于将 N 端 his 标签插入 pET3a-pro-MMP-3cd 载体
pET 质粒	Addgene	n/a	pET3a 载体提供氨苄青霉素抗性、靶基因的诱导表达以及使用 T7 引物进行测序
培养皿	VWR	25384-342	用于在 LB 琼脂培养基上接种转化体
R2DP 细胞	Novagen	714033	BL21 衍生物，真核蛋白表达增强。含有在大肠杆菌中发现罕见的密码子的 tRNA，SOC
生长培养基	NEB	B9020S	非选择性生长培养基，用于转化过程中的快速生长
氯化钠 （NaCl）	Thermo Fisher Scientific	BP358-1	用于缓冲液，有助于蛋白质稳定性
Thermo	Fisher Scientific	PI89905	用于蛋白质提取的去污剂。裂解细胞壁
增溶缓冲液	n/a	n/a	20 mM Tris-HCl （pH 8.0）、50 mM NaCl、10 mM DTT、6 M 尿素。将 pH 值调节至 8.0
Tris 碱	Thermo Fisher Scientific	BP152-1	常用缓冲液，用于生理 pH 值范围。温度敏感型
Triton X-100	Thermo Fisher Scientific	M1122980101	用于细胞裂解的去污剂
尿素	Thermo Fisher Scientific	AAJ75826A7	第一种用于破坏蛋白质二级结构的离液剂
氯化锌 （ZnCl2）	Thermo Fisher Scientific	AAA162810E	稳定 MMP 结构。锌离子存在于 MMP-3 的催化位点