Molecular Biology: A Practical Guide - Velikov V.A. 2013

Isolation of Recombinant Protein
Extraction of Protein from the Periplasm of E. coli Cells

To facilitate the subsequent Separation of the target protein from other E. coli Proteins, it can be directed into The Cell's periplasm immediately after synthesis on cytoplasmic Ribosomes is complete. The periplasmic space lies between the inner cytoplasmic membrane and The Cell wall (peptidoglycan layer). To achieve this, an appropriate vector is used during cloning. The genetic construct must contain nucleotide sequences encoding a specific leader peptide downstream of the LacZ promoter. An example is the leader of the pectate lyase Gene pelB from the bacterium Erwinia carotovora.

This leader is present in the pHEN1 phagemid (Hoogenboom et al., 1991). Introduction/27.html">Translation of the construct yields a covalently linked fusion protein (leader peptide - target protein), which accumulates in the cell's periplasm, thereby simplifying its separation from intracellular proteins without The Need for Chromatography. The leader peptide does not significantly affect The properties of the target protein. The accumulated protein is easily extracted from the cell periplasm, simply being released into solution upon suspending the Cells in a high-salt buffer. The borate method for isolating periplasmic proteins is described below.

Equipment and Materials

E. coli strain JS5 harboring the pHEN1 phagemid with the cloned scFv mini-antibody gene, high-speed centrifuge.

Solutions

- Borate buffer: 200 mM sodium borate, 60 mM NaCl, 1 mM EDTA.

- Protease inhibitor: PMSF or leupeptin, 1 mg/ml solution.

Procedure

1. Following the incubation period with the inducer, chill the cell culture on ice for 20 minutes to halt major biochemical processes and prevent the accumulation of incomplete synthesis products.

2. Pellet the ice-chilled cell culture by centrifugation at 10,000 rpm for 10 minutes at +4°C.

3. Discard the supernatant, and resuspend the cell pellet in 10 ml of ice-cold 200 mM sodium borate containing 160 mM NaCl and 1 mM EDTA by thorough pipetting. The high-salt buffer causes the cells to shrink, thereby squeezing the scFv out of the periplasm into the solution.

4. Remove the cells along with all cytoplasmic proteins by centrifugation at 4,000 rpm for 10 minutes. The supernatant will contain exclusively periplasmic proteins, with the target protein predominating.

5. Clarify the supernatant of cellular debris by centrifugation at 20,000 rpm for 30 minutes. Add a Serine protease inhibitor (PMSF or leupeptin) to the protein preparation at a ratio of 1 μl of stock solution per 1 ml of preparation.