Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence

Nandana, Vidhyadhar; Singh, Sushant; Singh, Abhay Narayan; Dubey, Vikash Kumar

doi:10.1016/j.pep.2014.08.003

Cited by 7 publications

(7 citation statements)

References 19 publications

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“…Mounting evidence suggests that the phenomenon of PS folding could be widespread 2,7,17–23 . Another interesting example are fold‐switching (or “metamorphic”) proteins, which have evolved to encode multiple, competing structures.…”

Section: Proteins In Nature Exist In a Wide Range Of Energy Landscapesmentioning

confidence: 99%

“…Pepsin and αLP remain the best‐characterized examples of PS folding, although the role of the PS during folding has been confirmed for several proteases across the major catalytic types 16 . This behavior has been reported in nearly 40 proteases, and it is likely more widespread than currently known based on conservation of PS motifs involved in the folding process 2,7,17–23 . It is important to clarify, however, that not all proteases require a PS to fold properly 8 .…”

Section: Introductionmentioning

confidence: 99%

“…16 This behavior has been reported in nearly 40 proteases, and it is likely more widespread than currently known based on conservation of PS motifs involved in the folding process. 2,7,[17][18][19][20][21][22][23] It is important to clarify, however, that not all proteases require a PS to fold properly. 8 Our understanding of protein frustration, 24 kinetic stability, 25 protein evolution, 26 and protein design principles 27,28 may be advanced by careful analysis of zymogen and mature protease folding.…”

Section: Introductionmentioning

confidence: 99%

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Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

2023

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In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha-lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self-assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non-cooperativity, memory effects, and substantial kinetic trapping.The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding "exceptions"-including protein fold switching, functional misfolding, and prevalent inability to refold-suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.

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Section: Proteins In Nature Exist In a Wide Range Of Energy Landscapesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

2023

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“…As the protein exhibited cysteine protease activity and thiol catalyzing hydrolysis, it could be categorized as a hydrolase, protease, and thiol protease; hence, the protease could be related to the milk-clotting property of D. sinensis and was the major milk-clotting protein. Nandana et al (2014) identified 127 AA in procerain B through N-terminal sequencing, and these AA belonged to the I29 inhibitory peptide. The tertiary structure was determined through x-ray crystal diffraction, and the recombinant active site of the enzyme was obtained by activating prokaryotic cells at pH 4.0 in vitro.…”

Section: Separation and Identification Of Proteases In D Sinensismentioning

confidence: 99%

Milk-clotting mechanism of Dregea sinensis Hemsl. protease

Zhang¹,

Wang²,

Tao

et al. 2015

Journal of Dairy Science

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Dregea sinensis Hemsl. is used as a milk coagulant to produce goat milk cakes in Yunnan, China. However, the composition of milk-clotting compounds and the related mechanism have not been reported. Crude protease was extracted from the stem, purified, and then separated with a Millipore ultrafiltration centrifuge tube. Cysteine protease (procerain B) was identified as the main milk-clotting protein through electrospray ionization mass spectrometry, and its molecular weight was 23.8 kDa. The protease can partially degrade α-casein (CN) and completely degrade β- and κ-CN, and κ-CN degradation resulted in milk clotting. The molecular weight and AA sequence of the peptide fractions were determined through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a peptide sequencer, respectively. The enzyme cleaved κ-CN at Ala90-Gln91 and produced deputy κ-CN and caseinomacropeptide with molecular weights of 12 and 6.9 kDa, respectively. This cleavage site differed from the majority of chymosins cleaved at Phe105-Met106.

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“…This pro-region probably serves a dual function, as both a folding template and an intrinsic inhibitor, preventing ectopic activation of the newly synthesized protein [ 11 – 13 ]. Therefore, the full-length cDNA information encoding full open reading frame (ORF) of enzyme is necessary for efficient expression of the proteases [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. Revealed by De Novo Transcriptome Analysis

et al. 2015

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Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

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Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence

Cited by 7 publications

References 19 publications

Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

Milk-clotting mechanism of Dregea sinensis Hemsl. protease

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. Revealed by De Novo Transcriptome Analysis

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