Expression of the cDNA Encoding the Pterocarpus angolensis (Mukwa Tree)-Seed Lectin in Escherichia coli and Site-Directed Mutagenesis of the Sugar-Binding Specificity Loop

Chidzwondo, Farisai (2005) Expression of the cDNA Encoding the Pterocarpus angolensis (Mukwa Tree)-Seed Lectin in Escherichia coli and Site-Directed Mutagenesis of the Sugar-Binding Specificity Loop. UNSPECIFIED thesis, UNSPECIFIED.

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The mannose/glucose specific lectin from Pterocarpus angolensis (mukwa tree) seeds was expressed in Escherichia coli using the pBAD expression system. The expression vector pBADMycHisA was digested with NcoI and filled-in with T4 DNA polymerase in order to introduce an initiator ATG codon preceding the polymerase chain reaction-amplified cDNA encoding the mature mukwa seed lectin. The recombinant plasmid was used to transform the expression cell line E. coli TOP10 cells. The cDNA clone, Muk151QII28, encoding the wild type mukwa seed lectin, was used as the template for oligonucleotide-directed mutagenesis of the sugar binding specificity. The first approach involved removing the part of the mukwa seed lectin sugar-specificity loop (loop D) that interacts with the sugar, and replacing it with the corresponding region of either the Ulex europaeus II lectin (UEA II) or the Erythrina corallodendron lectin (ECorL). In the second approach, two other mutants, predicted from X-ray crystallography to change the mukwa seed lectin sugar specificity from a-mannose/glucose to b-mannose/glucose, were generated. The DNA region carrying the mutations was then sub-cloned into the pBADMycHisA-wild type mukwa seed lectin recombinant in which the corresponding DNA region had been excised. The four mutants were expressed in E. coli TOP10 cells. The mutant lectins were assayed for cross-reactivity with antiserum directed against the native mukwa seed lectin in order to determine if the antiserum could be used in Western blotting. Hen egg white glycoproteins and glycoproteins of high variability isolated from porcine and bovine plasma were then blotted onto nitrocellulose and used to determine if the mutant lectins were capable of recognizing any carbohydrate moieties on glycoproteins. Maximum expression of both the wild type and the mutant lectins was obtained after induction with 0.2 % L-arabinose in cultures grown overnight. The presence or absence of a protease inhibitor cocktail did not seem to improve the yield. Up to 7.7 mg/500 ml culture of the expressed wild type lectin could be isolated from the extract by affinity chromatography on mannose-Sepharose. The purified lectin has a specific absorbance of OD280nm 1 mg/ml = 1.3 and shows an absorbance ratio of OD280nm/ OD250nm ≈3, the same as for the native lectin isolated from mukwa seeds. The expressed lectin has a slightly lower molecular mass than the native lectin but the two are essentially indistinguishable by Western blot analysis with anti-mukwa seed lectin polyclonal antibodies, haemagglutinating activity and both are inhibited by methyl-a-D-mannopyranoside. The mutant lectins cross-reacted with antiserum directed against the native mukwa seed lectin and all of them were capable of binding some carbohydrate moieties as shown by Western blotting. However, the wild type lectin showed a higher affinity for the carbohydrate moieties on the glycoproteins compared to the mutant lectins. The mutants, except for the UEA II specificity loop mutant, were successfully purified on an anti-mukwa seed lectin IgGSepharose column and used in agglutination assays. None of the mutants was capable of agglutinating any of the different animal erythrocytes tested showing that other factors apart from loop D determine sugar specificity in legume lectins.,Directorate General for International Cooperation, Flemish Interuniversity Council (DGIC/VLIR)

Item Type: Thesis (UNSPECIFIED)
Uncontrolled Keywords: Pterocarpus angolensis,mukwa seeds lectin,Escherichia coli,wild type lectin,mutant lectins,legume lectins
Divisions: Universities > State Universities > University of Zimbabwe
Depositing User: Mr. Edmore Sibanda
Date Deposited: 07 Dec 2015 01:31
Last Modified: 07 Dec 2015 01:31

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