Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinitychromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1)for the removal of C-terminusand N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidinetagofMBP-His6 by Factor Xa and HT 15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-HiS6 to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. Oncolumn tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-1 system was superiorto the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-1 system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-1 (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-1 digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-1 digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-l for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions

The penicillin-binding proteins of Bacilli

A study of the penicillin-binding proteins (PBPs) in the membranes of various species of Bacilli by dodecylsulphate/polyacrylamide gel electrophoresis followed by detection of the PBPs by fluorography has shown the presence of 3-5 such proteins depending on the species. Labelling of growing cells of Bacillus megaterium with [$^{14}C]benzylpenicillin showed that the PBP with the highest molecular weight, PBP1, was the only penicillin-binding protein that binds benzylpenicillin to any significant extent at the minimum growth inhibitory concentration. The covalent complexes formed between ~-lactams and the various penicillin-binding prdteins break down at different rates and the characteristics of the interaction of benzylpenicillin with PBP1 of B. megaterium are very similar to those of the inhibition by benzylpenicillin of the natural peptidoglycan transpeptidase reaction that is catalysed by cell-wall/ membrane preparations of this organism. PBP1 is proposed to be the peptidoglycan transpeptidase that represents the lethal target of penicillin action. Penicillin-binding proteins have been solubilized from the membranes with non-ionic detergents and isolated by affinity chromatography. Prior separation of the PBPs by ion-exchange chromatography followed by affinity chromatography has. resulted in the purification of PBPs 1, 3 and 4 of B. megaterium. PBP5 has also been purified by affinity chromatography and this purified protein catalyses a DD-carboxypeptidase reaction. None of the other purified PBPs has been shown to have enzymic activity in the terminal stages of peptidoglycan biosynthesis. The equivalent PBP1 from Bacillus licheniformis has been purified to ·protein homogeneity in large amounts in a single step process. Benzylpenicillin interacts with this purified protein to form a covalent stoicheiometric complex that subsequently breaks down and the half life of the complex is identical to that formed with the membrane-bound protein. The techniques used for the purification of PBP1 of B. megaterium have also been used to isolate PBP1 of Escherichia coli. The reactions that occur during the breakdown of benzylpenicillin-PBP complexes have been studied. The DD-carboxypeptidase of Bacillus stearothermophilus fragments benzylpenicillin to form phenylacetylglycine and N-formylD- penicillamine. The comparable reaction that occurs with PBP1 of B. licheniformis, either membrane-bound or in a purified form, appears to be more complex; bound benzylpenicillin is released via at least two different routes.Digitisation of this thesis was sponsored by Arcadia Fund, a charitable fund of Lisbet Rausing and Peter Baldwi