Engineering of Escherichia coli to improve the purification of periplasmic Fab' fragments: changing the pI of the chromosomally encoded PhoS/PstS protein.
Escherichia coli is a widely used host for the heterologous expression of proteins of therapeutic and commercial interest. The scale and speed at which it can be cultured can result in the rapid generation of large quantities of product. However, to achieve low costs of production a simple and robust purification process is also required. The general factors that impact on the cost of a purification process are the scale at which a process can be performed, the cost of the purification matrix, and the number and complexity of the chromatographic steps employed. Purification of Fab' fragments of antibodies from the periplasm of E. coli using ion exchange chromatography can result in the co-purification of E. coli host proteins having similar functional pI: such as the periplasmic phosphate binding protein, PhoS/PstS. In such circumstances, an additional chromatographic step is required to separate Fab' from PhoS. Here, we change the functional pI of the chromosomally encoded PhoS/PstS to effect its non-purification with Fab' fragments, enabling the removal of an entire chromatographic step. This exemplifies the strategy of the modification of host proteins with the aim of simplifying the production of heterologous proteins.
Humphreys
David P
DP
Celltech R+D, 216 Bath Road, Slough, Berkshire SL1 4EN, UK. david.humphreys@celltechgroup.com
Heywood
Sam P
SP
King
Lloyd M
LM
Bowering
Leigh C
LC
Turner
James P
JP
Lane
Sarah E
SE
D000818:Animals; D002845:Chromatography / Q000191:economics / Q000295:instrumentation / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007526:Isoelectric Point; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D028044:Phosphate-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D013499:Surface Properties
D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; C090133:PstS protein, E coli