Cloning and expression of the gene encoding a novel proteinase from Tritirachium album Limber

Samal, Babru; Karan, Barbara; Boone, Thomas C.; Chen, Kenneth K.; Rohde, Michael F.; Stabinsky, Y.

doi:10.1016/0378-1119(89)90425-3

Cited by 16 publications

(2 citation statements)

References 25 publications

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“…In the superfamily of trypsin-like serine proteases additional domains have also been added in several cases, both at the N-and C-terminal sides of the catalytic domain, through an evolutionary process called exon shuffling (Doolittle, 1985(Doolittle, , 1989Rogers, 1985;Irwin et a!., 1988). This evolutionary gene or domain coupling process may also apply to the subtilases, but the knowledge of gene intron -exon structure in this family is presently limited to HSFURI (van de Ven et a!., 1990), TAPROK (Gunkel and Gassen, 1989), TAPROT (Samal et a!., 1989) and AOALPR (Cheevadhanarah et al, 1991), and therefore insufficient for analysis of exon shuffling.…”

Section: Discussionmentioning

confidence: 99%

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

Siezen¹,

Vos²,

Leunissen

et al. 1991

Protein Eng Des Sel

348

293

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Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.

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Section: Discussionmentioning

confidence: 99%

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

Siezen¹,

Vos²,

Leunissen

et al. 1991

Protein Eng Des Sel

348

293

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“…The predicted Pen c 1 protein sequence shows extensive similarity with members of the subtilisin family of serine proteases (Fig. 4) and shares 51.4%, 48.2%, 46.4%, 44.7% or 35.1% identity of residues with Tritirachium album proteinase T [26], Metarhizium anisopliae Pr1 [27], Tritirachium album proteinase K [28], Aspergillus oryzae oryzin [29] and Bacillus subtilis subtilisin [30], respectively.…”

Section: Resultsmentioning

confidence: 99%

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum

Shen

et al. 1999

European Journal of Biochemistry

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A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

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