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

Summary

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.

Abstract

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.

Introduction

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 lead....

Protocol

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 ....

Results

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.......

Discussion

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.......

Materials

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