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Expression and characterisation of recombinant heparinase I expressed in Escherichia coli

James, Shelley (2021) Expression and characterisation of recombinant heparinase I expressed in Escherichia coli. Masters thesis, University of Sunderland.

Item Type: Thesis (Masters)

Abstract

Heparin is a complex, sulphated polysaccharide which is essential for many biological activities, one of which is to prevent blood coagulation. Because of its immediate effect, heparin has long been the parenteral drug of choice when a rapid anticoagulant effect is needed, particularly in the treatment of thromboembolism and deep vein thrombosis. However, blood samples contaminated with heparin are known to affect the Prothrombin Time and the Activated Partial Thromboplastin Time in in vitro tests. It is therefore imperative to be able to halt heparin activity when desired. Heparinases produced by Pedobacter heparinus neutralise the effects of heparin and allow for the unhindered analysis of haemostasis. P. heparinus produces three variants of heparinase, namely I, II and III, of which heparinase I is the most effective at neutralising heparin. Heparinase I has many clinical applications, which has led to the demand for a pure, high yield product. However, purifying HepI has proven to be time consuming, laborious, and can result in a low yield product. HepI has been expressed by others with solubility enhancing fusion partners, it has not however been produced in a yield that is commercially viable. The leading commercial provider of HepI, IBEX Technologies, produce the enzyme in 250-500 μl aliquots with a specific activity of 90-110 IU/mg. Initially, we aimed to show that soluble recombinant HepI protein could be expressed in high yield, which was just as, if not more, biologically active against unfractionated heparin and low molecular weight heparin than the leading commercial provider. Method and Results: A library of seven expression vectors was created to maximise soluble expression of HepI in Escherichia coli. DNA concentration and purity of each expression vector was determined by UV spectrophotometry at 260 nm and 280 nm wavelengths. All the expression plasmids DNA sequences were verified by Sanger sequencing and the amino acid sequence matched to the native HepI sequence when using a pairwise alignment tool. The concentration of the expression plasmids varied between 34.8 ng/μl and 126.5 ng/μl and purity ranged from 1.79 to 1.85. The plasmids were used to transform five host E. coli strains under IPTG induction and auto-induction, as well as with two induction temperatures. The concentration of soluble HepI protein expressed ranged from 9.8 to 17.1 mg per 35 ml culture. Five vector / host combinations were chosen for small scale purification using metal IMAC beads. The vector / host combination which produced the highest yield of purified HepI protein was pNTHIS-SUMOCDOHepI + BL21 (DE3) at 161.9 mg, however activity was found to be around 16 times lower than reported in the literature. Insoluble expression and refolding trials were conducted and resulted in the expression of 280 mg/L of insoluble HepI, with a specific activity calculated between 1876.38 and 2514.98 U/L culture across three
iv
experiments. Conclusion: Promising data were generated when expressing HepI in the insoluble form and results were comparable to the literature. Refolding trials produced between 1876.38 and 2514.98 U/L culture. Several avenues of optimising the process could be looked at such as new vector/host combinations, the use of a protamine anchor to increase Hep I affinity to heparin, utilising alternative expression hosts to aid in glycosylation and different methods of long-term storage such as lyophilising to reduce freeze-thaw denaturation.

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Depositing User: Unnamed user with email leaona.clarkson@sunderland.ac.uk

Identifiers

Item ID: 15056
URI: http://sure.sunderland.ac.uk/id/eprint/15056

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Catalogue record

Date Deposited: 15 Aug 2022 17:36
Last Modified: 15 Aug 2022 17:45

Contributors

Author: Shelley James

University Divisions

Faculty of Health Sciences and Wellbeing

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