1 Workflow Analysis of the Protein Purification Process of SeMet Labeled Proteins September 30, 2005 Haleema Janjua

2 Day One Two Step Purification Using AKTAxpress Day Two SDS-PAGE of fractions Collected by AKTA Day Three -Pool desired fractions -Concentrate -Aliquot Day Four Final SDS-PAGE and Mass Spec Day Five Bulk Upload Aggreg. Screening (Ken)

3 1 Equilibrate AKTAxpress 2 Obtain necessary info for each protein 3 5 Sonicate cell suspension (Total) Day One 7 Filter supernatent (0.45  m) (Soluble) 8 Load sample onto AKTAxpress and run overnight 4 Resuspend pellet in Binding Buffer Get pellet from freezer 6 Obtain supernatent by centrifugation

4 Day Two Analyze chromatograph and decide which fractions to run for SDS-PAGE Decide which fractions to pool based on result of chromatograph and SDS- PAGE Maintenance of AKTAxpress

5 Low expression/low solubility: ferment 2-4 liters Aggregation/precipitation: no salt buffer GF-AKTA or manual purification –need to develop further Planned Solution Low expression level (<2?) Low solubility (<2?) Aggregation or Precipitation in current buffer condition?

6 Day Three Pool fractions Based Unicorn Result Concentrate Sample to 5-10mg/ml By Amicon Ultrafree 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 -80  C (Flash frozen in LN2) ml in Eppendorf tube for SDS-PAGE and Mass spec (placed at 4  C) 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

7 Day Four Final SDS-PAGE Gel For Purity Mass Spec To Confirm MW Proteins with MW greater than or less than 500 Daltons from MW reported in Expression ID are held and submitted for LC-MS analysis (Peter Lobel’s group)

8 Day Five SDS-PAGE pictures taken Information Placed in SPINs In JPEG format Information Uploaded onto SPiNE Aggregation Screening information sent to Rong and Ken by Excel spreadsheet

9 Purifiy Selected Proteins using AKTA 133 Proteins Successfully purified Purification Flowchart Failed purification 44 Proteins (33%) 89 Proteins (67%) No binding 4 (9%) (New Construct) Low Sol 21 (48%) Low Exp/Low Sol 11 (25%) Low Exp 8 (18%) Precip Upon conc. (also failed AS) 8 (9%) Missed Peak 1 (1%) Low Yield (less than 5mg/L) 8 (9%) REFERMENT

10 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