The design, synthesis and expression of human platelet derived phospholipase A2 in Escherichia coli
The design, synthesis and expression of human platelet derived phospholipase A2 in Escherichia coli
A synthetic gene coding for the PLA2 derived from human platelet and also found enriched in rheumatoid arthritic synovial fluid has been designed, synthesised and expressed in Escherichia coli. A number of tightly controlled expression vectors were used in an attempt to overexpress this important enzyme. In the most successful expression system, the PLA2 gene was cloned under the control of a T7 promoter present on the pET11 plasmid. The system allowed the production of approximately 8mg per litre of PLA2 after purification. In order to produce PLA2 without the initiating methionine residue, which has only ~ 1% of the enzyme activity of the native human synovial fluid PLA2, a mutant was expressed with an alanine substituting the asparagine residue at position 1. This N1A mutant allowed the E. coli methionyl-aminopeptidase to cleave the initiator methionine while the resultant recombinant protein was shown to have the same specific activity and substrate specificity as native PLA2.
Further site-directed mutagenesis was performed on amino-terminal residues V3 and M8 of the N1A mutant and expressed using the T7 RNA polymerase vector, pET-11, in E. coli. This enabled production of 5-15mg per litre of these mutant enzymes. These were purified to >95% homogeneity and the activities of these mutants were evaluated. All the mutants showed the same substrate specificity for DOPG but had somewhat reduced enzyme activity which was most apparent with the M8N mutation. All the mutants gave similar rates for active site alkylation as the native PLA2 using para-bromophenacyl bromide. Only the M8N mutant was more unstable as judged from the heat stability experiments.
University of Southampton
1994
Othman, Roohaida
(1994)
The design, synthesis and expression of human platelet derived phospholipase A2 in Escherichia coli.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A synthetic gene coding for the PLA2 derived from human platelet and also found enriched in rheumatoid arthritic synovial fluid has been designed, synthesised and expressed in Escherichia coli. A number of tightly controlled expression vectors were used in an attempt to overexpress this important enzyme. In the most successful expression system, the PLA2 gene was cloned under the control of a T7 promoter present on the pET11 plasmid. The system allowed the production of approximately 8mg per litre of PLA2 after purification. In order to produce PLA2 without the initiating methionine residue, which has only ~ 1% of the enzyme activity of the native human synovial fluid PLA2, a mutant was expressed with an alanine substituting the asparagine residue at position 1. This N1A mutant allowed the E. coli methionyl-aminopeptidase to cleave the initiator methionine while the resultant recombinant protein was shown to have the same specific activity and substrate specificity as native PLA2.
Further site-directed mutagenesis was performed on amino-terminal residues V3 and M8 of the N1A mutant and expressed using the T7 RNA polymerase vector, pET-11, in E. coli. This enabled production of 5-15mg per litre of these mutant enzymes. These were purified to >95% homogeneity and the activities of these mutants were evaluated. All the mutants showed the same substrate specificity for DOPG but had somewhat reduced enzyme activity which was most apparent with the M8N mutation. All the mutants gave similar rates for active site alkylation as the native PLA2 using para-bromophenacyl bromide. Only the M8N mutant was more unstable as judged from the heat stability experiments.
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Published date: 1994
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Local EPrints ID: 458472
URI: http://eprints.soton.ac.uk/id/eprint/458472
PURE UUID: d1b9b896-f4da-41ee-99e0-f50ad972b09b
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Date deposited: 04 Jul 2022 16:49
Last modified: 04 Jul 2022 16:49
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Author:
Roohaida Othman
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