Targeted selection of amino acid residues to create variant libraries of Glaciozyma antarctica proline iminopeptidase

2019 ◽  
Author(s):  
Rohaiza Ahmad Redzuan ◽  
Nor Muhammad Mahadi ◽  
Abdul Munir Abdul Murad ◽  
Shazilah Kamaruddin ◽  
Farah Diba Abu Bakar
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Proline Iminopeptidase ◽  
Selection Of

Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

2019 ◽  
Vol 8 (1) ◽  
pp. 24-31
Author(s):  
Chol-Jin Kim ◽  
Sunll Choe ◽  
Kum-Chol Ri ◽  
Chol-Ho Kim ◽  
Hyon-Gwang Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Phage Display ◽  
Affinity Purification ◽  
Phage Library ◽  
Amino Acid Residues ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Selection Of

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

scholarly journals Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.

1991 ◽  
Vol 173 (3) ◽  
pp. 665-672 ◽  
Author(s):  
S Fish ◽  
M Fleming ◽  
J Sharon ◽  
T Manser
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Somatic Mutation ◽  
Amino Acid Substitution ◽  
Variable Region ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
V Region ◽  
Mutant Forms ◽  
Selection Of

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.

scholarly journals Probe design for mining and selection of genes coding endo 1- 4 xylanase from dna metagenome data

2018 ◽  
Vol 40 (1) ◽  
pp. 39-50
Author(s):  
Do Thi Huyen ◽  
Nguyen Minh Giang ◽  
Nguyen Thu Nguyet ◽  
Truong Nam Hai
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Probe Design ◽  
Amino Acid Residues ◽  
High Similarity ◽  
Metagenomic Dna ◽  
Free Living ◽  
Conserved Residues ◽  
Living Bacteria ◽  
Selection Of

According to the CAZY classification, endo 1- 4 xylanase belongs to GH 5, 8, 10, 11, 30, 51, 98. However only 03 sequences of GH8, 27 sequences of GH10, 18 sequence of GH11, only one sequence of each GH30 and GH51 from CAZy and NCBI database were thouroughly experimentally studied for biological activity and characteristics of the enzyme. Through the collected sequences, two probes for endo 1- 4 xylanase of GH10 and GH11 were designed, based on the sequence homology. The GH10 probe was 338 amino acids lenghth contained all the conserved amino acid residues (16 conserved residues in all sequences, 13 residues similar in almost sequences, 14 residues conserved in many sequences) with the lowest maxscore of 189, coverage of 88% and identity of 39%. The GH11 probe was 204 amino acids contained all the conserved amino acid residues (54 conserved residues were identity in all sequences, 25 residues similar in almost sequences, 24 residues conserved in many sequences) with the lowest maxscore of 165, coverage of 84% and identity of 50%. Using the two probes, we mined only one sequence (GL0018509) for endo 1- 4 xylanase from metagenomic DNA data of free-living bacteria in Coptotermes termite gut. Prediction of three-dimention structure of GL0018509 sequence by Phyre2 and Swiss Prot showed that this sequence was high similarity (95% by Phyre2 and 93,4% by Swiss Prot) with endo 1- 4 xylanase with the 100% confidence.

scholarly journals Substrate Range and Genetic Analysis ofAcinetobacter Vanillate Demethylase

2000 ◽  
Vol 182 (5) ◽  
pp. 1383-1389 ◽  
Author(s):  
Birgit Morawski ◽  
Ana Segura ◽  
L. Nicholas Ornston
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Nucleotide Substitutions ◽  
Two Component ◽  
Selection For ◽  
Type Sequence ◽  
Selection Of

ABSTRACT An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogsm-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-typeAcinetobacter bacteria. PCR mutagenesis ofvanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.

scholarly journals Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity

2013 ◽  
Vol 03 (03) ◽  
pp. 288-294 ◽  
Author(s):  
Kangkang Xing ◽  
Hong Feng
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Proline Iminopeptidase

scholarly journals Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs

2014 ◽  
Vol 95 (12) ◽  
pp. 2710-2715 ◽  
Author(s):  
Lizhen Wang ◽  
Yani Sun ◽  
Taofeng Du ◽  
Chengbao Wang ◽  
Shuqi Xiao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Amino Acid Residues ◽  
Antigenic Domains ◽  
Avian Hepatitis E Virus ◽  
Avian Hev ◽  
Selection Of ◽  
Antigenic Domain

The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

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