Abstract
We developed streamlined, automated purification protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) from Escherichia coli, using the ÄKTAxpress™ chromatography system. The automated 2-step (cation exchange and size exclusion) purification protocol for untagged hCypA results in final purity and yields of ⩾93% and ∼5mgL−1 of original cell culture, respectively, in under 12h, including all primary sample processing and column equilibration steps. The novel automated 4-step (anion exchange, desalt, heparin-affinity and size exclusion, in linear sequence) purification protocol for untagged hPCNA results in final purity and yields of ⩾87% and ∼4mgL−1 of original cell culture, respectively, in under 24h, including all primary sample processing and column equilibration steps. This saves in excess of four full working days when compared to the traditional protocol, producing protein with similar final yield, purity and activity. Furthermore, it limits a time-dependent protein aggregation, a problem with the traditional protocol that results in a loss of final yield. Both automated protocols were developed to use generic commercially available pre-packed columns and automatically prepared minimal buffers, designed to eliminate user and system variations, maximize run reproducibility, standardize yield and purity between batches, increase throughput and reduce user input to a minimum. Both protocols represent robust generic methods for the automated production of untagged hCypA and hPCNA.
Highlights
The demand placed on protein production strategies, in terms of the amount and purity of the protein products produced and the need to develop reliable and robust generic purification protocols, has increased massively in the post genome-sequencing era [1]
We developed reliable and robust automated purification protocols, for the production of milligram amounts of very pure untagged recombinant human CypA, by adapting an existing protocol [42], and for untagged human PCNA, by development of a novel 4-step protocol, using the ÄKTAxpressTM liquid chromatography system
Cell extract from up to 3 l of original E. coli culture could be processed without any apparent loss of purity or saturation of the dynamic binding capacity of the column ($40–60 mg protein mlÀ1 of wet resin).human cyclophilin-A (hCypA) invariably eluted between 13% and 22% Buffer-B and, following SDS–PAGE analysis, fractions collected between 14% and 22% Buffer-B were pooled and processed over a HiPrep 26/60 S-200 HR gel-filtration column (GE Healthcare), loading with a 10 ml loop with an additional 3 ml flush volume and run in Buffer-C (Table 1). hCypA invariably eluted as a symmetrical peak with an elution volume of 223.2 ± 0.3 ml
Summary
The demand placed on protein production strategies, in terms of the amount and purity of the protein products produced and the need to develop reliable and robust generic purification protocols, has increased massively in the post genome-sequencing era [1]. This is especially so for the increasing pace and scope of structural genomics and drug discovery programs, where there is very often a need to generate tens of milligrams of highly pure protein on a regular basis, with as little batch variation as possible [1,2,3,4,5].
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