Refolding Record:
| Protein | |
|---|---|
| Protein Name | Gelatinase B also known as Matrix metalloproteinase-9 & 92 kDa gelatinase |
| Abbreviated Name | MMP-9 |
| SCOP Family | MMP N-terminal domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P14780 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | amino acid residues 113-450 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 330 |
| Molecular Weight | 36459.3 |
| Pi | 4.78 |
| Molecular Weight | 36459.3 |
| Disulphides | Unknown |
| Full Sequence |
ITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTRDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kröger M, Tschesche H. (1997) Gene, 196, 175-180 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal methionine |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET12b |
| Expression Protocol | The cDNA that encodes C-terminally truncated 92-kDa gelatinase (amino acid residues 113–450) was cloned downstream from the bacteriophage T7 gene 10 promoter in the expression vector pET-12b, as shown in Fig. 2. The resulting plasmid 92KaDoGelpET-12b was used to transform E. coli strain BL21 (DE3). Induction of the expression system by adding IPTG yielded a recombinant protein which was deposited in its denatured form in inclusion bodies. Thirty minutes after induction, SDS–PAGE analysis indicated expression of a protein with an apparent molecular mass of approx. 40 kDa, as shown in Fig. 3A. Four hours after induction the amount of expression product was about 15% of total bacterial protein. Western blot analysis with antibodies against 92-kDa gelatinase showed cross-reactivity with the recombinant protein. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Other |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | redox-shuffling system in the presence of Zn2+ and Ca2+ |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Essential for the final yield of recombinant enzyme was the effective solubilization of the inclusion body fraction in 8 M urea. Contaminating E. coli proteins were eliminated by a combination of affinity chromatography and gel filtration. Purified short form 92-kDa gelatinase was obtained. Fig. 3B demonstrates the purification of the recombinant protein via two steps to apparent homogeneity. No impurity could be detected by silver staining. Preparation of eukaryotic proteins in E. coli often requires concomitant in vitro refolding. Renaturation of the denatured enzyme to active 92-kDa gelatinase was achieved after purification in a redox-shuffling system in the presence of Zn2+ and Ca2+. Under these conditions the recombinant protein remained soluble. Formation of insoluble aggregates during the renaturation process could be avoided by very low protein concentrations. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |