Expression Of Matrix Metalloproteinase26in Prokaryotic System And Characterization Its Enzymatic Properties

Matrix metalloproteinases (MMPs) are enzymes involved in extracellular matrixdegradation, which is a fundamental step in many physiological processes like tissueremodeling and repair. They have been almost exclusively studied in vertebrates and25paralogs are present in humans. Most MMPs are constituted by a prodomain that isremoved upon activation, a catalytic domain responsible for hydrolytic activity, and ahemopexin-like domain that probably plays a role in substrate recognition. In severalpathologies overexpression of MMPs, or the misregulation of their activity, is relatedto disease progression, such as arthritis, cardiovascular diseases, and cancer. MMPsthus are validated pharmaceutical targets. The main therapeutic strategy to combat thedysregulation of MMPs is the design of drugs to target their catalytic domains, forwhich purpose detailed structural knowledge is essential. The inability of first-andsecond-generation inhibitors to distinguish between different MMPs led to failures inclinical trials. More recent approaches have produced highly specific inhibitors totackle selected MMPs, thus anticipating more detailed structural characterization ofthe remaining MMPs to assist in achieving higher drug selectivity.MMP-26is a novel member of the matrix metalloproteinase family and initiallycloned from a human endometrial tumor cDNA library. The gene of MMP-26islocated at the11p15.3locus. It is the smallest MMP member with261amino acidresidues spanning, a signal domain, a propeptide domain and a catalytic domain.MMP-26lacks the hemopexin-like domai. It has a unique cysteine swith sequence(PH81CGVPDGSD) in the propeptide domain that contains a histidine residue insteadof the usual arginine residue. This unique cysteine switch leads to the unconventionalautolyitc activation of the MMP-26zymogen. MMP26and MMP7are the two smallest members of this family, exhibiting thesame domain structure constitute. They are have different roles in the biologicalfunction. MMP26cleaves multiple components of ECM, including fibronectin (FN),type IV collagen, vitronectin, gelatins and fibrinogen, as well as nonECM proteinssuch as insulinlike growth factor binding protein1(IGFBP1) and α1proteaseinhibitor. MMP26could activate proMMP9, thereby facilitates the efficient cleavageof ECM components and promotes the invasion of highly invasive and metastaticandrogenrepressed prostate cancer (ARCaP) cells. These results suggest that MMP26may possess critical roles in the processes of tumor invasion, angiogenesis, andmetastasis. MMP26mRNA has been observed in multiple cancers of epithelial origin,such as endometrial carcinoma, prostate carcinoma, lung carcinoma, theircorresponding cell lines, and in a malignant choriocarcinoma cell line (JEG3).However, the expression of MMP26mRNA is rare in normal adult tissues, andlargely limited to the uterus, placenta and kidney. MMP26expression is significantlyelevated in cancerous tissues of the human prostate when compared with prostatitis,benign prostate hyperplasia, and normal prostate tissues. Interestingly, the expressionof MMP26in human breast ductal carcinoma in situ (DCIS) is much higher than thatobserved in infiltrating ductal carcinoma (IDC), atypical intraductal hyperplasia, andnormal breast epithelia adjacent to DCIS and IDC. These results suggest that MMP26is involved in the initial invasion of human breast DCIS, and in the spread of prostatecarcinoma into surrounding stroma.Because the research of MMP-26is in the early stages, its physiological functionis still unknown. It is also a unique membrane of the MMPs family. Thethree-dimensional (3D) structure of the enzyme also remains to be deciphered.Previous studies show that MMP-26could be expressed in E. coli as inclusion bodiesand re-natured with catalytic activity. However, the low renaturation rate of thismethod limits its usage in structural studies, only5-15%of the enzyme would berefolded correctly with enzymatic activity. This method restricts the refolded proteinto be the material for structural research.Up to now, there are still many problems about expression of MMP-26in E. coli. Such as, if the catatlytic domain of MMP-26was expressed alone, refolded inactiveprotease would be possible. Some studies reported that propeptide of MMP-26wouldplay a chaperone role in helping its catalytic domain folded correctly, in theexpressing and refolding process. In addition, the pro form of MMP-26will not beactivated with other reagents or enzyme. It would undergo self-activation process withpropeptide. Because the process could not be controlled, acquiring enough activeMMP-26is relatively more complicated.MMP-26could be expressed in soluble form with enough quantity in prokaryoticsystem? If a large number of MMP-26could be successfully expressed in solubleform and folded correctly, with the enzymatic activity, the door would be opened forstructural studies. There is not a good expression system for recombinant MMP-26,from the MMP-26gene found in2000to now.In the current study, based on previous research, we explored soluble expressionof MMP-26in prokaryotic expression systems. Pro form and catalytic form ofMMP-26are small protein, with28kDa and19kDa molecular weight respectively.Because MMP-26have been expressed in E. coli and renatured into the active form,the prokaryotic expression system could be as the host for expressing the active formof MMP-26. This thesis will be commenced focusing on expression of MMP-26inprokaryotic system.First, we tried fusing ProMMP-26and CatMMP-26to several tags andexpressing the recombinant proteins in different E. coli strains. These tags were thehexahistidine (6×His) tag, maltose-binding protein (MBP) tag, thioredoxin (Trx) tag,ubiquitin (Ub) tag, small ubiquitin-related modifier (SUMO) tag and elastin-likepolypeptide-intein (EI) tag. We chose four E. coli strains for MMP-26expression,including BL21(DE3)，Rosetta(DE3)，Rosetta-gami2(DE3) and BLR(DE3). Thegrowth and induction conditions were optimized for expression of the MMP-26recombinant protein. We selected the soluble form for studying. Cell lystates fromun-induced (UI), and induced (I) cultures, plus soluble (S) and inclusion bodies (IB)fraction from the induced cultures were analyzed by SDS–PAGE. When recombinant MMP-26fused with tags, including6×His， Trx，SUMO and UBIQUITIN, theseconstructs gave little soluble recombinant MMP-26expression in the E. coli strains(BL21(DE3), Rosetta(DE3), Rosetta-gami2(DE3)) and most of the MMP-26proteinswere expressed as inclusion bodies. MBP-ProMMP-26/MBPCatMMP-26wereexpressed in the E. coli and the soluble form of the protein as well as high expressionlevels were obtained in Rosetta (DE3). We tried to remove the MBP tag by thrombin.However, the fusion protein MBP-ProMMP-26or MBP-CatMMP-26could not beefficiently cleaved by thrombin as only partial cleavage products were obtained,suggesting that MBP-ProMMP-26/CatMMP-26may have formed soluble aggregatesduring expression. Moreover, the cleaved product showed no enzymatic activitiesindicating that the protein was not correctly folded. EI-ProMMP-26/EI-CatMMP-26were also expressed in the soluble fraction in E. coli BLR (DE3), but the expressionlevel was significantly low. EI-ProMMP-26/EI-CatMMP-26purification processcould not be conducted by using the property of ELP-INTEIN tag. In fact,ProMMP-26/CatMMP-26with MBP and EI tags could expressed as soluble forms inE. coli strains. However, the expressed soluble recombinant protein did not adopt thecorrect conformation.We next resorted to express recombinant MMP-26in the Gram-positive bacteriaB. choshinensis system. This organism contains a naturally efficient secretion systemto direct expressed proteins into the culture supernatant, which often results inimproved yield and ease of purification. The B. choshinensis system is nearly free ofproteases, which facilitates production of intact protein products. It also facilitatesdisulfide bond formation. For certain proteins, its expression system is superior to E.coli. In order to express recombinant MMP-26in B. choshinensis, the B.choshinensis-E. coli shuttle vectors of pNCMO2-His6-ProMMP-26andpNCMO2-CatMMP-26-His6were constructed by inserting the cDNA sequences ofProMMP-26and CatMMP-26into pNCMO2plasmid with6×His tag inserted at N-and C-termini respectively. After optimizing secreted expression conditions bydifferent culture conditions, we have successfully expressed ProMMP-26andCatMMP-26in soluble form. Purified His6-ProMMP-26have gelatin zymography activity, would undergo auto-activation throughout the purification process, thisphenomenon is more obvious as the purity of the protein increases and when theprotein is more concentrated. Purified CatMMP-26-His6has high enzymatic activityagainst DQ-gelatin substrate and gelatin zymography activity.Our work has removed the obstacles to express MMP-26in native conformationusing the prokaryotic system and it will facilitate further structural determination ofthis unique matrix metalloproteinase. After storage of purified catalytic domain ofMMP-26(CatMMP-26-His6) for some time, it would take place the hydrolysis. So weexpect to acquire the stable active form MMP-26.Next we have constructed four kinds of the catalytic domain of MMP-26expression plasmids, including pHis6-CatMMP-26，pHis6-CatMMP-26ΔEKCSSDIP+KN， pHis6-CatMMP-26ΔTSISPGRC，pHis6-CatMMP-26E209A. These constructs were purified by the6×His tag in theN-terminus. They are the wild type, N terminal deletion, C terminal deletion andconserved active-site motif point mutations. These catalytic domain of MMP-26constructs were successfully secreted expressed in B. choshinensis system. They werein stable form after purification. We found that His6-CatMMP-26，His6-CatMMP-26ΔEKCSSDIP+KN and His6-CatMMP-26ΔTSISPGRC hadenzymatic activity against DQ-gelatin substrate and gelatin zymography activity.Enzymatic activity against DQ-gelatin substrate of His6-CatMMP-26andHis6-CatMMP-26ΔTSISPGRC were fairly the same level with CatMMP-26-His6,while the enzymatic activity of His6-CatMMP-26ΔEKCSSDIP+KN decreased.These results suggested that the C-terminal MMP-26non-conserved amino acidsplay a role of molecular chaperone, in a stable conformation with them, once thecompletion of protein folding they could be removed without affecting the enzymaticactivity. Non-conserved amino acids in the N-terminal of CatMMP-26did not affectthe conserved active-site motif and protein stability. Cys97and Cys256did not interferethe conformation of conserved active-site motif, so as not to affect its enzymaticactivity.In conclusion, in this study we have successfully expressed ProMMP-26and CatMMP-26in soluble form in the B. choshinensis system, they are able to be foldedcorrectly. Meanwhile we constructed the deletion and point mutations of CatMMP-26,revealing relationship between the enzymatic activity of CatMMP-26with the proteinstability.