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PubMed1994

Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Amino Acid Sequence↗
PubMedDate not supplied

Construction of prostate-specific expressed recombinant plasmids with high transcriptional activity of prostate-specific membrane antigen (PSMA) promoter/enhancer.

To screen different combinations of prostate-specific membrane antigen (PSMA) promoter/enhancer with the strongest transcriptional activity in prostate-specific cells, we used PSMA regulatory elements to control specific expression of the target gene in gene therapy of prostate adenocarcinoma. PSMA promoter and enhancer DNA sequences were amplified from the LNCaP human prostate cancer cell line by polymerase chain reaction, then recombinant plasmids of the enhanced green fluorescent protein (EGFP: pEGFP-PSMA(Pro), pEGFP-PSMA(E-P), pEGFP-PSMA(E(r)-P), pEGFP-PSMA(E(d)-P), and pEGFP-PSMA(E(t)-P)) were constructed with molecular clonal techniques. At the same time, all experimental cell lines were analyzed for the expression of PSMA with the use of PSMA monoclonal antibody and the ABC immunohistochemical assay kit. After plasmids were transfected via liposome, we observed the expression of the reporter gene (EGFP) under a fluorescent microscope and compared the different levels of EGFP expression with reverse transcriptase polymerase chain reaction and flow cytometry so that we could choose the one with the highest transcriptional activity. Only the LNCaP cell line expressed PSMA positively with immunohistochemical stain. The PSMA promoter/enhancer had transcriptional activity in PSMA(+) cell lines and no activity in PSMA(-) cell lines. PSMA(E-P) achieved the strongest activity in different PSMA promoter/enhancer combinations. We confirmed the specific expression of PSMA in prostate cells again. Similarly, transcriptional activity of the PSMA promoter/enhancer was prostate specific. PSMA(E-P) achieved the strongest transcriptional activity among PSMA promoter/enhancer combinations, which could be used in advanced research for tissue-specific treatment.

Antigens, Surface↗
PubMed2004

Recombinant expression of mutations causing von Willebrand disease type Normandy: characterization of a combined defect of factor VIII binding and multimerization.

Von Willebrand disease type Normandy (VWD 2N) is caused by mutations at the factor VIII (FVIII) binding site of VWF, located at the amino-terminus of mature VWF. It is inherited in a recessive fashion and both homozygous and compound heterozygous mutations have been identified. Homozygous mutations are correlated with a clinical phenotype indistinguishable from mild hemophilia A by conventional laboratory tests, whereas compound heterozygosity with a quantitative defect may appear as VWD type 1 (VWD1). We have now identified and expressed a novel heterozygous mutation (Y795C) which is responsible for both, a defective FVIII-binding and aberrant multimers in a female patient with mild FVIII deficiency. Additionally we expressed another mutation (E787K), previously identified by us in a male patient with a severe 'pseudohemophilic' phenotype. Analysis of the FVIII binding and the multimer structure of the respective recombinant VWF mutants reproduced the observed phenotype: the FVIII binding defect in addition to the aberrant multimer structure of the patient with Y795C and the FVIII binding defect only, in the patient with E787K. Our results demonstrate the causative nature of the two mutations and emphasize the impact of 'cysteine mutations' on the multimer structure of VWF.

Adult↗
PubMed2001

Leishmania donovani: expression and characterization of Escherichia coli-expressed recombinant chitinase LdCHT1.

Leishmania parasites produce chitinase activity (EC. 3.2.1.14) thought to be important in parasite-sandfly interactions and transmission of the parasite to the vertebrate host. Previous observations have suggested that parasite chitinases are involved in degradation of the sandfly peritrophic matrix and the chitinous layer of the cardiac valve cuticle. This chitinase activity is thought to produce an incompetent pharyngeal valve sphincter and a route of egress that allow Leishmania promastigotes to be regurgitated into the site of blood feeding. In the studies reported here, enzymatically active L. donovani chitinase LdCHT1 was expressed as a thioredoxin fusion protein in Escherichia coli strain AD494 (DE3). Recombinant LdCHT1 had a predominantly endochitinase activity, in contrast to previous reports of both exo- and endochitinase activities in axenic culture supernatants of diverse Leishmania spp. promastigotes. The predominant endochitinase activity of recombinant LdCHT1 is consistent with the presumed function of the enzyme in disrupting chitinous structures in the sandfly digestive system to allow transmission.

Acetylglucosamine↗
PubMed1999

Selective induction of apoptosis by the cytotoxic analog AN-207 in cells expressing recombinant receptor for luteinizing hormone-releasing hormone.

The selectivity of ligands specific for certain cells can be used to preferentially target chemotherapeutic compounds to neoplastic cells. Human breast, ovarian, endometrial, and prostatic cancers express receptors that can mediate the delivery of targeted cytotoxic compounds to neoplastic cells. Recently, a potent derivative of 2-pyrrolinodoxorubicin (AN-201) conjugated to [D-Lys6] luteinizing hormone-releasing hormone (LH-RH) (AN-207), was demonstrated to be less toxic than the nonconjugated chemotherapeutic radical and significantly more active in slowing neoplastic cellular growth. In this study we investigate the molecular mechanisms underlying the cytotoxic action of AN-207. We stably transfected COS cells with a LH-RH receptor (LH-RH-Rc) mammalian expression vector and examined the effect of AN-207 on known markers of cellular apoptosis. Apoptotic induction by AN-207, as measured by Bax and Bcl-2 protein levels, was increased in stable cells that express LH-RH-Rc compared with parental cells. DNA fragmentation also was increased by AN-207 treatment when compared with AN-201. Clinically used LH-RH antagonists partially inhibited apoptotic Bax expression and DNA fragmentation induced by AN-207, and blocked AN-207 induced down-regulation of Bcl-2 steady-state protein levels. In cell proliferation studies, after 72 h AN-207 exhibited greater cytotoxicity than AN-201 at equivalent concentrations, in COS cells expressing LH-RH-Rc but not in parental COS cells. In addition, survival of LH-RH-Rc positive cells treated with AN-207 was partially restored by LH-RH antagonist. This study demonstrates the receptor-specific cytotoxic effect of 2-pyrrolinodoxorubicin conjugated to [D-Lys6] LH-RH, exerted through induction of apoptosis and modulation of Bax, Bcl-2, and DNA fragmentation.

Animals↗
PubMed2004

Recombinant expression, purification and cross-reactivity of chenopod profilin: rChe a 2 as a good marker for profilin sensitization.

Chenopod pollen is one of the major sources of allergens in some locations in the US, southern Europe and desert countries, and pollen profilin (Che a 2) is a major allergen. Recombinant Che a 2 (rChe a 2) has been produced in Escherichia coil cells with a final yield of 25 mg/l of cell culture. The expressed protein was isolated and structurally characterized by means of mass spectrometry, Edman degradation and circular dichroism. rChe a 2 displayed a molecular mass of 13 959 Da, which agrees with that of the amino acid sequence. The N-terminal amino acid sequence indicated the correct processing of the recombinant product. The immunological analysis of rChe a 2 showed IgG- and IgE-binding capabilities equivalent to those of its natural counterpart, Che a 2, isolated from the pollen. Inhibition experiments showed high cross-reactivity degrees with different allergenic sources. Inhibition degrees of >95% and >80% were obtained for chenopod profilin and, respectively, latex and pollen extracts, whereas 10-95% of inhibition was observed for different plant-derived foods. Due to its close relation to other allergenic profilins from pollens, plant-derived foods and latex, rChe a 2 could be a useful tool in clinical trials to detect profilin-allergic patients and perhaps, depending on its clinical relevance, in specific immunotherapy of these hypersensitive individuals.

Allergens↗
PubMed1997

Cloning, sequencing, and recombinant expression of NAT1, NAT2, and NAT3 derived from the C3H/HeJ (rapid) and A/HeJ (slow) acetylator inbred mouse: functional characterization of the activation and deactivation of aromatic amine carcinogens.

An acetylator polymorphism has been described in the mouse and the inbred strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, respectively. The NAT1, NAT2, and NAT3 genes from C3H/HeJ and A/HeJ acetylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced. They were then subcloned into the prokaryotic expression vector pKK223-3 and expressed in Escherichia coli strain JM105. The 870-bp nucleotide coding region of NAT1 and NAT3 did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouse NAT1 and NAT3 sequences. However, NAT2 did differ between the rapid and slow acetylator strains with an A296 T transition which causes a (Asn99-->Ile) substitution in the deduced amino acid sequence. Recombinant NAT1, NAT2, and NAT3 proteins catalyzed N-, O-, and N,O-acetyltransferase activities. NAT3 catalyzed aromatic amine N-acetyltransferase activities at very low rates, which confirms a previous study. Apparent K(m) and Vmax kinetic constants for N-acetylation were 5- to 10-fold lower for recombinant mouse NAT1 than NAT2. Intrinsic clearances for recombinant mouse NAT1- and NAT2-catalyzed N-acetylation of aromatic amine carcinogens were comparable. Both recombinant mouse NAT1 and NAT2 catalyzed the metabolic activation of N-hydroxyarylamine (O-acetylation) and N-hydroxyarylamide (N,O-acetylation) carcinogens. Recombinant mouse NAT3 catalyzed N,O-acetylation at very low rates, while O-acetylation was undetectable. No difference was observed between rapid and slow acetylator recombinant NAT2 proteins to activate aromatic amines by O- or N,O-acetylation, in substrate specificity, expression of immunoreactive protein, electrophoretic mobility, or N-acetyltransferase Michaelis-Menten kinetic constants. However, the slow acetylator recombinant NAT2 protein was over 10-fold less stable than rapid acetylator recombinant NAT2. These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2, and NAT3 proteins and confirm and extend previous studies on the molecular basis for the acetylation polymorphism in the mouse.

Acetylation↗
PubMed2000

The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange.

An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced. The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively. To the best of our knowledge, this is the highest directly measured melting temperature of a native protein. Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites. Obviously it is not the incorporation of a metal ion that confers the extreme thermostability. Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.

Amino Acid Sequence↗
PubMed2001

Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability.

Although Candida rugosa utilizes a nonuniversal serine codon (CUG) for leucine, it is possible to express lipase genes (LIP) in heterologous systems. After replacing the 19 CUG codons in LIP4 with serine codons by site-directed mutagenesis, a recombinant LIP4 was functionally overexpressed in Pichia pastoris in this study. This recombinant glycosylated lipase was secreted into the culture medium with a high purity of 100 mg/liter in a culture broth. Purified recombinant LIP4 had a molecular mass of 60 kDa, showing a range similar to that of lipase in a commercial preparation. Since LIP4 has only a glycosylation site at position Asn-351, this position may also be the major glycosylation site in C. rugosa lipases. Although the thermal stability of recombinant LIP4 significantly increased from 52 to 58 degrees C after glycosylation, there were no significant differences in the catalytic properties of recombinant glycosylated lipase from P. pastoris and the unglycosylated one from Escherichia coil. These two recombinant LIP4s showed higher esterase activities toward long-chain ester (C16 and C18) and exhibited higher lipase activities toward unsaturated and long-chain lipids. In addition, LIP4 does not show interfacial activation as compared with LIP1 toward lipid substrates of tributyrin and triolein. These observations demonstrated that LIP4 shows distinguished catalytic activities with LIP1 in spite of their high sequence homology.

Candida↗
PubMedDate not supplied

Mutation in the Escherichia coli htpR locus results in stabilization of recombinant expression products that are susceptible to proteolytic degradation.

We compared the expression and degradation of three cloned malarial proteins in a pair of isogeneic strains of Escherichia coli that differed at the htpR locus. The htpR locus encodes an alternate sigma factor necessary for the transcription of heat shock promoters. Plasmodium sequences were cloned from polymerase chain reaction-amplified DNA initiated by oligonucleotide primers that were specific for the gene coding regions to be expressed. The amplified DNA was cloned and expressed in a vector that encodes a strong T7 promoter and translation--initiation signal. The total cell yield of two of the expressed proteins was found to be increased when synthesis occurred in a E. coli htpR mutant. Pulse--chase experiments showed that the increased protein yield correlated with a decrease in the degradation of the protein in the htpR strain. A two- to seven-fold increase in the half-life of the malaria proteins was observed in the E. coli htpR- background as compared to htpR+. We found no difference in survival of the E. coli K165 htpR mutant and isogeneic parent during thermal induction. Since the synthesis of the heat shock sigma factor did not significantly influence survival of E. coli and htpR expression results in increased degradation of foreign proteins, the E. coli htpR mutant was a valuable host strain for production of foreign proteins.

Amino Acid Sequence↗
PubMed1997

Recombinant expression of caveolin-1 in oncogenically transformed cells abrogates anchorage-independent growth.

Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype. Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.

3T3 Cells↗
PubMed2003

Recombinant expression of the Ca(2+)-sensitive aspartate/glutamate carrier increases mitochondrial ATP production in agonist-stimulated Chinese hamster ovary cells.

The Ca(2+)-sensitive dehydrogenases of the mitochondrial matrix are, so far, the only known effectors to allow Ca2+ signals to couple the activation of plasma membrane receptors to the stimulation of aerobic metabolism. In this study, we demonstrate a novel mechanism, based on Ca(2+)-sensitive metabolite carriers of the inner membrane. We expressed in Chinese hamster ovary cells aralar1 and citrin, aspartate/glutamate exchangers that have Ca(2+)-binding sites in their sequence, and measured mitochondrial Ca2+ and ATP levels as well as cytosolic Ca2+ concentration with targeted recombinant probes. The increase in mitochondrial ATP levels caused by cell stimulation with Ca(2+)-mobilizing agonists was markedly larger in cells expressing aralar and citrin (but not truncated mutants lacking the Ca(2+)-binding site) than in control cells. Conversely, the cytosolic and the mitochondrial Ca2+ signals were the same in control cells and cells expressing the different aralar1 and citrin variants, thus ruling out an indirect effect through the Ca(2+)-sensitive dehydrogenases. Together, these data show that the decoding of Ca2+ signals in mitochondria depends on the coordinate activity of mitochondrial enzymes and carriers, which may thus represent useful pharmacological targets in this process of major pathophysiological interest.

Adenosine Triphosphate↗
PubMed1996

Development of drug targeting based on recombinant expression of the chicken avidin gene.

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.

Animals↗
PubMed1995

Recombinant expression of the brush border myosin I heavy chain.

Although the specific functions of myosin I motors are not known, their localization to membrane structures suggests a function in membrane motility. Different myosin I isoforms in the same cell or in different cells can possess different localizations. To determine if the localization and biochemical activity of the best-characterized mammalian myosin I, chicken intestinal epithelium brush border myosin I, was dependent on determinants of the membrane or actin cytoskeleton specific to epithelial cells, we transfected the cDNA for the heavy chain of this myosin into COS cells. Transient transfection of COS cells with the chicken brush border myosin heavy chain resulted in the production of recombinant myosin I. Recombinant brush border myosin I localized to protrusions of the plasma membrane, particularly at spreading edges, and also to unknown cytoplasmic structures. Some cells expressing particularly high levels of brush border myosin I possessed a highly irregular surface. Recombinant brush border myosin I purified from COS cells bound to actin filaments in an ATP-dependent manner and decorated actin filaments to form a characteristic appearance. The recombinant myosin also catalyzed calcium-sensitive, actin-activated MgATPase activity similar to that of the native enzyme. Thus, any cellular factor required for the general membrane localization or biochemical activity of brush border myosin I is present in COS cells as well as intestinal epithelium.

Actins↗
PubMed2006

Ribavirin uptake by cultured human choriocarcinoma (BeWo) cells and Xenopus laevis oocytes expressing recombinant plasma membrane human nucleoside transporters.

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.

Animals↗
PubMed2000

Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.

Animals↗
PubMed1996

Mengo virus 3C proteinase: recombinant expression, intergenus substrate cleavage and localization in vivo.

Mengo virus 3C proteinase was cloned and expressed to high levels in a bacterial vector system. The protein was solubilized from inclusion bodies then purified to homogeneity (> 95%) by ion exchange chromatography. The recombinant enzyme was proteolytically active in cell-free processing assays with a Mengo capsid precursor substrate, L-P1-2A, correctly and proficiently cleaving it into L, 1AB, 1C, 1D and 2A protein products. Further analyses with synthetic peptide substrates encompassing the Mengo or rhinovirus-14 2C/3A cleavage sequences, showed the Mengo 3C could recognize and process specific glutamine-glycine sites within these peptides. The reactivity with the rhinovirus peptide was unexpected, because cross-reactivity between a picornavirus 3C enzyme and a protein substrate from different genus of this family has otherwise never been observed. In reciprocal reactions, a rhinovirus-14 3C preparation was unable to cleave the Mengo-derived synthetic peptide substrate. The recombinant Mengo 3C reactions were also characterized with regard to substrate Km, optimum pH and temperature. The protein was additionally used to raise monoclonal antibodies (mAbs) in mice, which in turn localized natural 3C, 3ABC, 3CD and P3 in immunoblots, immunoprecipitations and indirect immunofluorescence assays of Mengo-infected HeLa cells. The monoclonals showed cross-reactivity with 3C and 3C-containing precursors from encephalomyocarditis virus (EMCV), but did not react with 3C proteins from rhinovirus-14 or poliovirus-1M.

3C Viral Proteases↗