A Study On Purification And Refolding Of MMP-13 By Chromatography

Matrix metalloproteinases (MMPs) are a family of extracellular zinc-dependent neutral endopeptidases collectively capable of precisely regulating degradation of the extracellular matrix (ECM). The development of many diseases relates to the abnormal expression of MMPs. MMPs are reported to have significant effect on many diseases such as rheumatoid arthritis, cancer and cardiovascular diseases. Therefore, taking MMPs as the target for inhibitor screening has became an important approach for the therapy of many diseases, especially cancer. This paper describes the research of induction expression, chromatographic purification and chromatographic refolding of MMP-13, MMP-13 inhibitor screen and preparation of MMP-13-inhibitor complexes, that is the basis for the study of inhibitory site and inhibition mechanisms.The main contents of this study include:1. ReviewIn this section, the progress at home and abroad in studies of cancer treatment strategies and anti-cancer drugs, matrix metalloproteinases and inhibitors, protein refolding methods, protein structure research was summarized.2. MMP-13 expression in vitroRecombinant E.coli was used as expression system, and recombinant MMP-13 catalytic domain protein was induced and expressed by IPTG. In addition, by employing LB medium or M9 medium, we found that the protein expression yield with LB medium or M9 medium was 41.3% and 39.8%, respectively.3. Purification of MMP-13The protein mixture was obtained through the treatment process including ultrasonication, centrifuging, inclusion body washing, inclusion body dissolution. Protein was purified by Ni2+-affinity chromatography, and its purity was increased from 75.2% to 92.6%, and the mass yield was increased from 60.7% to 72.8% after the optimization of some conditions, such as the concentration of imidazole:15 mmol/L, loading volume:1.20 mL (protein concentration,18.93 mg/mL), and flow rate:1.00 mL/min.4. Refolding of MMP-13Ion exchange chromatography and size exclusion chromatography were used to refold MMP-13. Ion exchange chromatography performed with three gradient was first used for refolding with the mass yield of 84.2% when loading volume was 0.40 mL (protein concentration was 0.9360 mg/mL), flow rate was 0.80 mL/min, elution gradient time was 25 min, and pH of B buffer was 7.0. And then size exclusion chromatography was used for desalting with the mass yield of 21.3%. The activity of refolded MMP-13 was 1.188×105 mF.U./mn (positive control was 1.209×105 mF.U./mn). About 18.6 mg/L of active protein was obtained. Using the other refolding method, size exclusion chromatography, the mass yield was 31.7%, activity was 0.9916×105 mF.U./mn (positive control was 1.209x 105 mF.U./mn), and about 32.8 mg/L of active protein was obtained.5. Screening of MMP-13 inhibitors and the effect on purification and refoldingK3[Fe(CN)6] was found to have the best activity (IC5o is 1.7μmol/L) by testing the inhibitory activity of A12(SO4)3, FeCl3, K3[Fe(CN)6], etc to MMP-13. The yield of protein was increased by 30% due to the effect of K3[Fe(CN)6] on preparation of inclusion bodies, purification and refolding of MMP-13.6. The preparation MMP-13-inhibitorsTaking Al2(SO4)3,K3[Fe(CN)6] as inhibitors, three protein samples (MMP-13 was renatured without inhibitors, MMP-13-inhibitor complex means that inhibitors were added through purification and refolding, and MMP-13-inhibitor complex means that inhibitors were added after purification and refolding) were prepared for analyzing the structure of MMP-13-inhibitors and revealing the inhibitory site by X-ray crystallography and 3D NMR.