Journal Article

Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins

Kao, Louis T., Barrett, Lynn K., Hewitt, Stephen N., Nguyen, Trang Nhu, Van Voorhis, Wesley C., Leibly, David J.

2012

Overview

Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2), proline, xylitol, NDSB 201, CTAB and K(2)PO(4)) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Additives / Arginine / Betaine / Biochemistry / Biology / Buffers / Cell Fractionation / Chemistry / Citric acid / Copper chloride / Crystallization / Crystallography / Detergents / E coli / Electrophoresis, Polyacrylamide Gel / Enzymology / Escherichia coli / Excipients - chemistry / Excipients - pharmacology / Genomics / Glycine / Glycine betaine / Health screening / In vivo methods and tests / Inclusion bodies / Infectious diseases / Ligands / Lysis / Mannitol / Medicine / Models, Biological / Potassium / Proline / Protein expression / Proteins / Protozoan Proteins - isolation & purification / Recombinant proteins / Recombinant Proteins - metabolism / Salts / Screening / Solubility / Solubility - drug effects / Solubilization / Trehalose / VOCs / Volatile organic compounds / Xylitol