In-process Monitoring of Fusion Protein

The following experiments demonstrate use of the SILAS surface to monitor affinity purification of a recombinant fusion protein. The assays are 5 minutes long and the information provides for quick evaluation of the efficiency of column binding and elution. The assays take advantage of the high capacity protein adsorption properties of the SILAS surface. Under the proper buffer conditions the silicon surface binds protein avidly. Adding serum or Blockaid reagent to the diluent controls nonspecific binding of detector antibody. The basic steps in protein purification evaluation are:

Step 1: Bind protein mixture to SILAS surface: 1 minute

Step 2: Detect protein of interest with specific antibody-HRP conjugate plus substrate: 4 minutes

The SILAS surfaces were used to monitor efficiency of purification of a glutathione S-transferase-Neisseria gonorrhea (GST-GC) fusion protein. The recombinant protein was expressed in E. coli and purified from the cell lysate by affinity purification with a glutathione column (Amersham Pharmacia Biotech). The basic steps in this purification scheme (adapted from Amersham Pharmcia Biotech) were:

1. Induce expression of fusion protein and prepare lysate.
2. Apply lysate to glutathione affinity column, elute with glutathione. Expect GST-GC fusion in eluate.
3. Cleave fusion with serine protease (GST + GC).
4. Apply cleaved preparation to glutathione column, elute with glutathione. Expect GC protein in flow through, GST in eluate.

Fractions from the flow-through and eluate were evaluated with the SILAS surface in a 5 minute assay format. The fusion protein was detected with antibody to the fusion protein partners, either anti-GC antibody HRP conjugate or anti-GST antibody HRP conjugate.

Protocol

1. Dilute column fraction 1:5 in Protein Diluent.
2. Add 20 µl of diluted fraction to individual SILAS chips, incubate 1 minute.
3. Wash surface with wash or phosphate buffer and blot dry.
4. Add 20 µl anti-species HRP conjugate to surface, incubate 2 minutes.
5. Wash surface with buffer and blot dry.
6. Add TMB and incubate for 2 minutes.
7. Wash surface with buffer and blot dry.

A. GST-GC fusion protein purification with the glutathione affinity column, eluted with free glutathione

Expect GST-GC fusion protein in eluate

The thin film biosensor quickly revealed a significant amount of fusion protein in the flow-through, indicating column overloading or insufficient binding conditions. Obtaining this data while purification is in progress allows for protocol adjustment, re-purification of the flow through, and identification of eluate fractions to pool for proteolytic cleavage.

B. Evaluation of Post Protease Cleavage Purification

• The purified GST-GC fusion was cleaved with serine protease to separate the fusion partners.
• The protein was applied to the glutathione column and eluted with free glutathione.
• GC protein was expected in the flow-through, GST in the eluate
• Fractions were evaluated in-process with the SILAS rapid assay

Results demonstrate purification of GC protein in the flow-through, with residual uncleaved fusion protein in these fractions. The reactivity of the eluted fractions also suggests insufficient protease cleavage, resulting in GC protein in the eluate, still linked to GST as a fusion protein.

The data from the SILAS assay quickly identify which fractions to consider for repeated protease treatment and purification.