Sloane, Rhone Patricia; (1996) Construction, expression and purification of a histidine-tailed bacteriophage T4 lysozyme. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Many recombinant proteins are expressed in the periplasm of Gram negative bacteria. Release of these proteins can be achieved by the use of muramidases but these are expensive and can act as potential process contaminants further downstream. A version of bacteriophage T4 lysozyme has been constructed by the addition of a His-Gln-(His)3 peptide to the C-terminus of a cysteine-free protein. The expressed fusion protein can be purified using immobilized metal affinity chromatography and reused. The protein was initially recovered (to a high level) on iminodiacetic acid (IDA) Sepharose columns charged with Zn2+, Ni2+, and Cu2+ from crude cell lysates. Since no binding to the columns was observed with the wild type protein, the interaction can be attributed to the fusion tail. The retention strength on the columns was Cu>Ni>Zn, although few differences in the levels of purity and recovery of the enzyme were observed. In addition, the purification of the same recombinant protein from crude and clarified cell extracts using novel non-porous ferromagnetic supports (coated with IDA and M2+) is described. Various parameters were investigated in order to achieve maximal recovery of purified product while maintaining enzyme activity. The capacity of Cu-charged supports was determined for both extracts (0.35 mg/ml clarified, 0.45 mg/ml crude), the buffer which provided maximal binding was investigated (20 mM sodium phosphate, 0.2 M NaCl, pH 7.2) as was that which effected maximal elution (0.1 M sodium acetate, 0.5 M NaCl, pH 3.5). It is recommended that two wash steps would eliminate unbound proteins prior to elution of the target protein where greater than 90% was recovered in two steps. The minimum time required for maximal binding under the conditions used here was 5 minutes. Other divalent metal ions were tested for their ability to bind the target protein and only Cu, Ni and Zn exhibited any significant success. The order of retention was as found in the column chromatography - Cu>Ni>Zn. Recovery and purity levels were similar to those obtained on the columns. Consecutive recycling without cleansing treatment is not recommended above two cycles.

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