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Workflow Analysis of the Protein

Purification Processof SeMet Labeled Proteins

September 30, 2005Haleema Janjua

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Day OneTwo Step Purification

Using AKTAxpress

Day TwoSDS-PAGE of fractions

Collected by AKTA

Day Three-Pool desired fractions

-Concentrate-Aliquot

Day FourFinal SDS-PAGE

andMass Spec

Day FiveBulk Upload

Aggreg. Screening(Ken)

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1Equilibrate AKTAxpress

1Equilibrate AKTAxpress

2Obtain necessary info

for each protein

2Obtain necessary info

for each protein

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5Sonicate cell suspension

(Total)

5Sonicate cell suspension

(Total)

Day One

7Filter supernatent (0.45m)

(Soluble)

7Filter supernatent (0.45m)

(Soluble)

8Load sample onto

AKTAxpress and runovernight

8Load sample onto

AKTAxpress and runovernight

4Resuspend pellet in

Binding Buffer

4Resuspend pellet in

Binding Buffer

Get pellet from freezer

6Obtain supernatent by

centrifugation

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Day Two

Analyze chromatographand decide which fractions to run

for SDS-PAGE

Decide which fractions to pool based on result of chromatograph and SDS-PAGE

Maintenance of AKTAxpress

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•Low expression/low solubility: ferment 2-4 liters•Aggregation/precipitation: no salt buffer GF-AKTA or manual purification –need to develop further

PlannedSolution

Low expression level (<2?) Low solubility (<2?)

Aggregation or Precipitation in current buffer condition?

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Day Three

Pool fractions Based Unicorn

Result

Pool fractions Based Unicorn

Result

Concentrate Sample to 5-10mg/mlBy AmiconUltrafree

Device

Concentrate Sample to 5-10mg/mlBy AmiconUltrafree

Device

Determine Concentration at 280nm by Diluting protein with 6M Guanadine+ 10mM Tris, pH 7.5

-Upload purification record to SPiNE and obtain PST IDs

-Aliquot proteins in the following manner:1. 0.4ml in 1.7ml Eppendorf tube for HWI

(Flash frozen in LN2)2. 0.1ml in vial for Aggregation Screening

(Flash frozen in LN2)3. 1.6ml in PCR strips (50ul/tube) for

Columbia (Flash frozen in LN2)4. Leftover stored at -80C (Flash frozen in

LN2)5. 0.02ml in Eppendorf tube for SDS-PAGE

and Mass spec (placed at 4C)Note: In case of volume less than 0.5ml ask to

referment 2 or more liters

1. In case of precipitation

2. Stop further concentrating

3. Remove precipitate by centrifugation

4. Analyze supernatent

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Day Four

Final SDS-PAGE

GelFor Purity

Mass Spec To

Confirm MW

Proteins with MWgreater than or less than 500 Daltons from MW reported in Expression ID are held and submitted forLC-MS analysis (PeterLobel’s group)

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Day Five

SDS-PAGEpictures taken

InformationPlaced in SPINsIn JPEG format

InformationUploaded ontoSPiNE

Aggregation Screening informationsent to Rong and Ken byExcel spreadsheet

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Purifiy SelectedProteins using AKTA

133 ProteinsSuccessfully purified

Purification Flowchart

Failed purification

44 Proteins (33%) 89 Proteins (67%)

No binding4 (9%)

(New Construct)

Low Sol21 (48%)

Low Exp/Low Sol11 (25%)

Low Exp8 (18%)

Precip

Upon conc.

(also

failed AS)

8 (9%)

Missed Peak

1 (1%)

Low Yield(less than 5mg/L)

8 (9%)

REFERMENT

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Recommended Recovery

Failed Purification-make a new construct and purify again-Evaluate how ExS can be used to screen those samples that failed purification

Precipitation upon concentrating-Consider different buffer-Consider different Aggregation Screening buffer-Perform button test to figure out better conditions

Low yield-Scale up and repeat purification

Missed peak-Repeat fermentation and purify