Information on EC 3.4.23.B2 - Simian immunodeficiency virus proteinase

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The expected taxonomic range for this enzyme is: Simian immunodeficiency virus

EC NUMBER
COMMENTARY hide
3.4.23.B2
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
Simian immunodeficiency virus proteinase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
The enzyme may have a wide substrate specificity. Good cleavage of the peptide bonds Met-Met and Tyr-Pro. Cleavage is also observed at Phe-Pro, Phe-Leu, Leu-Phe, Leu-Ala, Glu-Ala and Tyr-Ala
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Ac-Ala-Thr-Leu-Asn-Phe-Pro-Ile-Ser-Pro-Ile-Glu-NH2 + H2O
Ac-Ala-Thr-Leu-Asn-Phe + Pro-Ile-Ser-Pro-Ile-Glu-NH2
show the reaction diagram
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-
-
?
Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 + H2O
Ac-Arg-Ala-Ser-Gln-Asn-Tyr-OH + Pro-Val-Val-NH2
show the reaction diagram
Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2 + H2O
Ac-Arg-Lys-Ile-Leu + Phe-Leu-Asp-Gly-NH2
show the reaction diagram
-
-
-
-
?
Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 + H2O
Ac-Ser-Gln-Asn-Tyr + Pro-Val-Val-NH2
show the reaction diagram
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-
-
?
aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-NH2 + H2O
?
show the reaction diagram
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-
-
-
?
biotin-Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala-Pro-Gln-Phe-Ser-Leu-Trp-Arg-Arg + H2O
biotin-Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala + Pro-Gln-Phe-Ser-Leu-Trp-Arg-Arg
show the reaction diagram
-
-
-
?
galactokinase + H2O
?
show the reaction diagram
Lys-Ala-Arg-Val-Nle-(NO2)Phe-Glu-Ala-Nle-Gly + H2O
Lys-Ala-Arg-Val-Nle + (NO2)Phe-Glu-Ala-Nle-Gly
show the reaction diagram
oxidized insulin B chain + H2O
?
show the reaction diagram
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cleavage sites: HLVE13-ALYL and EALY16-LVC[SO3-]G
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-
?
Pr55Gag polyprotein + H2O
?
show the reaction diagram
SIV Gag precursor protein + H2O
?
show the reaction diagram
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determination of cleavage sites and substrate specificity
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-
?
troponin C + H2O
?
show the reaction diagram
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cleavage sites: EEEL97-AECF and AEEL117-AEIF
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-
?
Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val + H2O
Val-Ser-Gln-Asn-Tyr + Pro-Ile-Val
show the reaction diagram
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?
additional information
?
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cleavage site in Lys-PE40, a construct based upon the Pseudomonas exotoxin OE66 that is missing domain I are: ANL-1-AEEA and GDAL389-LERN
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Pr55Gag polyprotein + H2O
?
show the reaction diagram
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lentiviral Gag polyprotein, proteolytic processing for activation, cleavage in the late stages of life cycle, required for maturation of the virion meaning a structural rearrangement required for infectivity in budding virions, cleavage in the phosphorylated capsid domains, the enzyme is not influenced by the preceeding phosporylation, but cleaved sites are no longer phosphorylated, while incompletely cleaved capsid domains are phosphorylated to a greater extent, overview
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
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1 M NaCl, rate of substrate cleavage increases up to 4fold
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,2-epoxy-3-(4-nitrophenoxy)propane
1-naphthoxyacetyl-His-cyclohexylacetyl-PSI[CH(OH)CH(OH)]Val-Ile-2-pyridylmethylamine
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9H-fluoren-9-ylmethyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate
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specific irreversible inhibition, t1/2 for inactivation is 240 min with a 0.1 mM inhibitor concentration
Ac-cyclohexylacetyl-PSI[CH(OH)CH2]Val-Glu-Arg-NH2
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Ala-Ala-Phe-Psi[CH(OH)CH2]Gly-Val-ValOCH3
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H-Val-Ser-Gln-Asn-Leu-PSI[CH(OH)CH2]Val-Ile-Val-OH
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pepstatin A
phenoxyacetyl-His-cyclohexylacetyl-PSI[CH(OH)CH(OH)]Phe-Ile-2-pyridylmethylamine
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phenoxyacetyl-His-Leu-PSI[CH(OH)CH2]Phe-Ile-2-pyridylmethylamine
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SFK 107457
SFK 108842
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IC50: 0.00013
SFK 108922
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IC50: below 0.000077 mM
SFK 109274
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IC50: above 0.02 mM
SFK 17461
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IC50: 0.0048 mM
SKF 108390
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IC50: 0.00063
SKF 108907
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IC50: 0.008 mM
SKF107457
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a hydroxyethylene inhibitor
U-85548
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i.e. Val-Ser-Gln-Asn-Leu-PSI[CH(OH)CH2]-Val-Ile-Val
ZnCl2
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1 mM, no detectable activity
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6
Ac-Ala-Thr-Leu-Asn-Phe-Pro-Ile-Ser-Pro-Ile-Glu-NH2
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pH 6, 37°C
3.1
Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val
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pH 6, 37°C
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0.3
Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2
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pH 6, 37°C
4.3
Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2
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pH 6, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.6
Ac-Ala-Thr-Leu-Asn-Phe-Pro-Ile-Ser-Pro-Ile-Glu-NH2
Simian immunodeficiency virus
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pH 6, 37°C
6.2
Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val
Simian immunodeficiency virus
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pH 6, 37°C
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1.3
Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2
Simian immunodeficiency virus
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pH 6, 37°C
6.4
Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2
Simian immunodeficiency virus
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pH 6, 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.023
1-naphthoxyacetyl-His-cyclohexylacetyl-PSI[CH(OH)CH(OH)]Val-Ile-2-pyridylmethylamine
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pH 5.5, 30°C
0.2
Ac-cyclohexylacetyl-PSI[CH(OH)CH2]Val-Glu-Arg-NH2
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pH 5.5, 30°C
0.000012
Ala-Ala-Phe-Psi[CH(OH)CH2]Gly-Val-ValOCH3
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pH 4.7, 37°C
0.3
amastatin
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pH 6, 37°C
0.3
chymostatin
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pH 6, 37°C
0.006
H-Val-Ser-Gln-Asn-Leu-PSI[CH(OH)CH2]Val-Ile-Val-OH
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pH 5.5, 30°C
0.003
pepstatin A
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pH 6, 37°C
0.49
phenoxyacetyl-His-cyclohexylacetyl-PSI[CH(OH)CH(OH)]Phe-Ile-2-pyridylmethylamine
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pH 5.5, 30°C
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0.19
phenoxyacetyl-His-Leu-PSI[CH(OH)CH2]Phe-Ile-2-pyridylmethylamine
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pH 5.5, 30°C
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additional information
additional information
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0102
SFK 107457
Simian immunodeficiency virus
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IC50: 0.0102 mM
0.00013
SFK 108842
Simian immunodeficiency virus
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IC50: 0.00013 mM
0.000077
SFK 108922
Simian immunodeficiency virus
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IC50: below 0.000077 mM
0.02
SFK 109274
Simian immunodeficiency virus
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IC50: above 0.02 mM
0.0048
SFK 17461
Simian immunodeficiency virus
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IC50: 0.0048 mM
0.00063
SKF 108390
Simian immunodeficiency virus
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IC50: 0.00063 mM
0.008
SKF 108907
Simian immunodeficiency virus
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IC50: 0.008 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 or Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly as substrate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 50
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20°C: 60% of maximal activity, 50°C: about 80% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.1
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calculation with the program PREDICT
7
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mutant enzyme K69H
8.2
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isoelectric focusing
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
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x * 10000, SDS-PAGE
10812
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2 * 10812, electrospray mass spectrometry
11600
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2 * 11600, SDS-PAGE
18000 - 19000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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the retropepsin is expressed as part of a larger polyprotein precursor, which it cleaves into functional proteins, the major autodegradation site of the enzyme is Phe3-Ser4
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
complex of the enzyme bound to the inhibitor SKF107457, hanging drop method of vapor diffusion
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crystal structure determination and analysis of free enzyme and enzyme-inhibitor complexes, e.g. with 1,2-epoxy-3-(4-nitrophenoxy)propane at 2.4 A resolution, or with SKF107457 at 2.5 A resolution
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enzyme covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane. Crystals of the variant SIV PR S4H appear to have the same morphology as those obtained from wild-type enzyme
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hanging drop method
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hanging-drop crystallization of the K69H mutant enzyme with pepstatin A
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SIV protease product complex, hanging drop vapor diffusion method
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unliganded SIV protease crystals are grown by hanging drop vapor diffusion. Domain flexibility and structural implications for drug resistant mutations
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 6
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60 min, stable
647815
7 - 8
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activity is slightly diminished, less than 30%, after preincubation at pH 7.0-8.0
647815
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
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thermal denaturation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
8 M urea cause 50% quenching of the fluorescence emission
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microcrystals are stable under mild acidic conditions
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purified enzyme is unstable due to autopropteolysis
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, 50 mM sodium acetate, pH 5.0, 5 mM EDTA, 10 mM DTT, 0.35 NaCl, 40% v/v glycerol
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-80°C, in 10% glycerol, no loss of activity after 12 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli by ammonium sulfate fractionation, and ion exchange or affinity chromatography on a pepstatin A resin, recombinant N-terminally capped, biotin-labeled precursor by streptavidin affinity chromatography, the tag is autocatalytically cleaved after refolding of the enzyme
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SIVmac251-32H proteinase from recombinant Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned directly from proviral DNA of the infectious viral stock SIVmac251-32H(11/88 pool). SIVmac251-32H proteinase and ist flanking pol sequences are expressed in Escherichia coli as a fusion protein with most of the T7 bacteriophage gene 10 protein. The expressed protein forms cytoplasmic inclusion bodies which are solubilized in 8 M urea, and the recombinant enzyme is refolded, yielding active self-processing enzyme
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expressed in Escherichia coli with a recombinant expression system
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expression in Escherichia coli
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expression in Escherichia coli, expression as N-terminally capped, biotin-labeled precursor
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D25N
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totally inactive mutant enzyme, incapable of autoprocessing
K69H
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the isoelectric point of the mutant enzyme is pH 7.0 compared to that of the wild-type enzyme of 7.4. The purified mutant enzyme is less soluble at low pH when concentrating sample in preparation for crystallization
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nearly total restoration of protein fluorescence upon cooling to 25°C of the enzyme which has been unfolded at 70°C
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refolding from recombinant cytoplasmic inclusion bodies, refolding of recombinant N-terminally capped, biotin-labeled precursor
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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SIV retropepsin can serve as an inhibitor-resistant retrovirus variant model of HIV-1 retropepsin