Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis

Khouri, Henry E.; Vernet, Thierry; Ménard, Robert; Parlati, Francesco; Laflamme, Pierre; Tessier, Daniel C.; Gour-Salin, Barbara J.; Thomas, David Y.; Storer, Andrew C.

doi:10.1021/bi00101a003

Cited by 84 publications

(49 citation statements)

References 42 publications

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“…This also needs enhancement of papain for the use in these media. From literature, it is known that this can be achieved by immobilization [54][55][56], modification with polyethylene glycol [57][58][59] or site directed mutagenesis [60].…”

Section: Discussionmentioning

confidence: 99%

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Schwab¹,

Kloosterman²,

Konieczny³

et al. 2012

Polymers

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Four hydrophobic amino acids (Leu, Tyr, Phe, Trp) were oligomerized by the protease papain in homo-oligomerization, binary co-oligomerization and ternary co-oligomerization. After 24 h, solid polydisperse reaction products of the homo-oligomerization were obtained in yields ranging from 30-80% by weight. A DP avg was calculated based on MALDI-ToF MS results using the ion counts for the chains in the product. Based on the DP avg and the yield of the homo-oligomerization it was determined that the amino acids can be ranked according to reactivity in the order: Tyr > Leu > Phe > Trp. Thermal degradation of the homo-oligomers shows two degradation steps: at 178-239 °C and at 300-330 °C. All the products left a significant amount of char ranging from 18-57% by weight at 800 °C. Binary co-oligomers were obtained as a polydisperse precipitate with a compositional distribution of the chains. Both the compositional and chain length distribution are calculated from MALDI-ToF mass spectra. By comparing the amount of each amino acid present in the chains it was determined that the amino acids are incorporated with a preference: Leu > Tyr > Phe > Trp. Ternary co-oligomers were also obtained as a precipitate and analyzed by MALDI-ToF MS. The compositional distribution and the chain length distribution were calculated from the MALDI-ToF data. The quantity of every amino acid in the chains was determined. Also determined was the influence on the DP avg when the oligomers were compared with corresponding binary co-oligomers. From the combined results it was concluded that in the co-oligomerization of three amino acids the reactivity preference is Leu > Tyr > Phe > Trp. OPEN ACCESSPolymers 2012, 4 711Thermal degradation of all the co-oligomers showed a weight loss of 2 wt% before the main oligomer degradation step at 300-325 °C.

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Section: Discussionmentioning

confidence: 99%

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Schwab¹,

Kloosterman²,

Konieczny³

et al. 2012

Polymers

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“…2). Site-directed mutagenesis of the gene for the papaya CP, papain has altered the engineered enzyme's preference for peptide substrates away from that of papain towards that of the mammalian CP cathepsin B (Khouri e t al., 1991).…”

Section: Discussionmentioning

confidence: 99%

Multiple high activity cysteine proteases of Leishmania mexicana are encoded by the Imcpb gene array

Robertson

Coombs

1994

Microbiology

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“…All buffers contained 1 mM EDTA and 0.4 M NaCl to minimize the variation in ionic strength. A three-protonation model (22) was used for least square regression analysis of the pH activity data. The data were fitted to the following equation.…”

Section: Construction Of a Pcdna31-based Expression Vector For Cathementioning

confidence: 99%

Human Cathepsin F

Wang

Shi

Yao

et al. 1998

Journal of Biological Chemistry

153

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A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a gt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.

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Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis

Cited by 84 publications

References 42 publications

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Multiple high activity cysteine proteases of Leishmania mexicana are encoded by the Imcpb gene array

Human Cathepsin F

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