Limits...
Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using AKTAxpress.

Ludwig C, Wear MA, Walkinshaw MD - Protein Expr. Purif. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Protein Production Facility, Centre for Translational and Chemical Biology, University of Edinburgh, Michael Swann Building, Edinburgh EH9 3JR, UK.

Show MeSH

Related in: MedlinePlus

Automated Purification of hPCNA. (A) Typical chromatogram for theautomated 4-step purification of hPCNA using ÄKTAXpress™. The pre-packedcolumns used are illustrated above the corresponding section of the chromatogram; IEX– ion-exchange, DS – desalt, AF – affinity, GF –gel-filtration. Solid black; A280 nm in mAU (left axis). Solid red; elution gradient in %Buffer-E or Buffer-G (right axis),IEX and AF step, respectively. The buffer pairs used are indicated above theappropriate portion of the chromatogram. The inset details the region of thegel-filtration column elution from which fractions were collected. Indicatedfractions A7–C11 were pooled. (B) SDS–polyacrylamide gel (4–20%gradient) illustrating the final purity levels of hPCNA purified by both manual(⩾93%) and automated (⩾87%) protocols (determined by gel densitometry). Thefinal purity from 2 independent traditional and 2 independent automatic runs areshown, illustrating the excellent reproducibility of both methods. Five μg total protein was loaded in each lane. SCE, soluble cell extract;R1, run 1; R2, run 2. Molecular weight markers are shown to the right of the gel.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this paper.)
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2837147&req=5

fig2: Automated Purification of hPCNA. (A) Typical chromatogram for theautomated 4-step purification of hPCNA using ÄKTAXpress™. The pre-packedcolumns used are illustrated above the corresponding section of the chromatogram; IEX– ion-exchange, DS – desalt, AF – affinity, GF –gel-filtration. Solid black; A280 nm in mAU (left axis). Solid red; elution gradient in %Buffer-E or Buffer-G (right axis),IEX and AF step, respectively. The buffer pairs used are indicated above theappropriate portion of the chromatogram. The inset details the region of thegel-filtration column elution from which fractions were collected. Indicatedfractions A7–C11 were pooled. (B) SDS–polyacrylamide gel (4–20%gradient) illustrating the final purity levels of hPCNA purified by both manual(⩾93%) and automated (⩾87%) protocols (determined by gel densitometry). Thefinal purity from 2 independent traditional and 2 independent automatic runs areshown, illustrating the excellent reproducibility of both methods. Five μg total protein was loaded in each lane. SCE, soluble cell extract;R1, run 1; R2, run 2. Molecular weight markers are shown to the right of the gel.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this paper.)

Mentions: Novel protocols for the purification of recombinant untagged hPCNA fromE. coli were developed from scratch (see Materials andMethods for full details). We first developed a traditional 4-step ion-exchange(IEX), desalt (DS), affinity (AF), gel-filtration (GF) methodology, againutilizing generic pre-packed columns (suitable for use on theÄKTAXpress™ system), and a set of automatically generated minimalbuffers (Buffer-D, Buffer-E,Buffer-F, Buffer-G, Table 1; Fig.2). The IEX column elution uses arelatively complex 4-step gradient profile: a wash immediately following sampleapplication with 15 column volumes of 27% Buffer-E,followed by a 2.7 column volume gradient from 27% to 54%Buffer-E, followed by a 5 column volume gradient from54% to 57% Buffer-E, followed by a step to 100%Buffer-E. Relevant hPCNA fractions from 50.7% to 55.5%Buffer-E were pooled and further processed. Thisgradient profile proved critical for ensuring hPCNA eluted with as narrow anelution peak as possible (between 16 and 17 ml), while at thesame time limiting the level and number of contaminants, allowing an easy 2-repeatrun processing through the subsequent desalting step without furtherconcentration. hPCNA purified by this method was ⩾93% pure as judged bydensitometric analysis of SDS–polyacrylamide gels (Fig. 2B) and typically yielded ∼2.5 mg per litre of original bacterial culture (Table 2). Despite the fact that very pure proteincould be reproducibly obtained (Fig.2B), the traditional method requires a very considerable amount ofmanual intervention and typically takes 5 full working days to process the proteinfrom cell pellet through to purity. Furthermore, a significant amount of protein(∼1–2 mg) was lost over the course of purificationdue to a time-dependent aggregation and resulting precipitation problems. Theseeffects were only partially alleviated by the addition of glycerol to thechromatography buffers. Thus, in an attempt to reduce the loss of protein, togreatly reduce the amount of user input to a minimum and to further standardizethe purification, we streamlined this method further by translating it into afully automated protocol on the ÄKTAXpress™ system.


Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using AKTAxpress.

Ludwig C, Wear MA, Walkinshaw MD - Protein Expr. Purif. (2009)

Automated Purification of hPCNA. (A) Typical chromatogram for theautomated 4-step purification of hPCNA using ÄKTAXpress™. The pre-packedcolumns used are illustrated above the corresponding section of the chromatogram; IEX– ion-exchange, DS – desalt, AF – affinity, GF –gel-filtration. Solid black; A280 nm in mAU (left axis). Solid red; elution gradient in %Buffer-E or Buffer-G (right axis),IEX and AF step, respectively. The buffer pairs used are indicated above theappropriate portion of the chromatogram. The inset details the region of thegel-filtration column elution from which fractions were collected. Indicatedfractions A7–C11 were pooled. (B) SDS–polyacrylamide gel (4–20%gradient) illustrating the final purity levels of hPCNA purified by both manual(⩾93%) and automated (⩾87%) protocols (determined by gel densitometry). Thefinal purity from 2 independent traditional and 2 independent automatic runs areshown, illustrating the excellent reproducibility of both methods. Five μg total protein was loaded in each lane. SCE, soluble cell extract;R1, run 1; R2, run 2. Molecular weight markers are shown to the right of the gel.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this paper.)
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837147&req=5

fig2: Automated Purification of hPCNA. (A) Typical chromatogram for theautomated 4-step purification of hPCNA using ÄKTAXpress™. The pre-packedcolumns used are illustrated above the corresponding section of the chromatogram; IEX– ion-exchange, DS – desalt, AF – affinity, GF –gel-filtration. Solid black; A280 nm in mAU (left axis). Solid red; elution gradient in %Buffer-E or Buffer-G (right axis),IEX and AF step, respectively. The buffer pairs used are indicated above theappropriate portion of the chromatogram. The inset details the region of thegel-filtration column elution from which fractions were collected. Indicatedfractions A7–C11 were pooled. (B) SDS–polyacrylamide gel (4–20%gradient) illustrating the final purity levels of hPCNA purified by both manual(⩾93%) and automated (⩾87%) protocols (determined by gel densitometry). Thefinal purity from 2 independent traditional and 2 independent automatic runs areshown, illustrating the excellent reproducibility of both methods. Five μg total protein was loaded in each lane. SCE, soluble cell extract;R1, run 1; R2, run 2. Molecular weight markers are shown to the right of the gel.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this paper.)
Mentions: Novel protocols for the purification of recombinant untagged hPCNA fromE. coli were developed from scratch (see Materials andMethods for full details). We first developed a traditional 4-step ion-exchange(IEX), desalt (DS), affinity (AF), gel-filtration (GF) methodology, againutilizing generic pre-packed columns (suitable for use on theÄKTAXpress™ system), and a set of automatically generated minimalbuffers (Buffer-D, Buffer-E,Buffer-F, Buffer-G, Table 1; Fig.2). The IEX column elution uses arelatively complex 4-step gradient profile: a wash immediately following sampleapplication with 15 column volumes of 27% Buffer-E,followed by a 2.7 column volume gradient from 27% to 54%Buffer-E, followed by a 5 column volume gradient from54% to 57% Buffer-E, followed by a step to 100%Buffer-E. Relevant hPCNA fractions from 50.7% to 55.5%Buffer-E were pooled and further processed. Thisgradient profile proved critical for ensuring hPCNA eluted with as narrow anelution peak as possible (between 16 and 17 ml), while at thesame time limiting the level and number of contaminants, allowing an easy 2-repeatrun processing through the subsequent desalting step without furtherconcentration. hPCNA purified by this method was ⩾93% pure as judged bydensitometric analysis of SDS–polyacrylamide gels (Fig. 2B) and typically yielded ∼2.5 mg per litre of original bacterial culture (Table 2). Despite the fact that very pure proteincould be reproducibly obtained (Fig.2B), the traditional method requires a very considerable amount ofmanual intervention and typically takes 5 full working days to process the proteinfrom cell pellet through to purity. Furthermore, a significant amount of protein(∼1–2 mg) was lost over the course of purificationdue to a time-dependent aggregation and resulting precipitation problems. Theseeffects were only partially alleviated by the addition of glycerol to thechromatography buffers. Thus, in an attempt to reduce the loss of protein, togreatly reduce the amount of user input to a minimum and to further standardizethe purification, we streamlined this method further by translating it into afully automated protocol on the ÄKTAXpress™ system.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Protein Production Facility, Centre for Translational and Chemical Biology, University of Edinburgh, Michael Swann Building, Edinburgh EH9 3JR, UK.

Show MeSH
Related in: MedlinePlus