Increase protein production yield at a considerably reduced cost with HYPE-5 transfection reagent

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.

J Vis Exp. 2015 Dec 23;(106). doi: 10.3791/53445.

Kober DL¹, Yurtsever Z², Brett TJ³.

This article demonstrates the high efficiency of HYPE-5 for protein production.

Abstract

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

"Optimal cell viability and transfection conditions are the most crucial for efficient protein production. For better cell viability, low-passage cell cultures are boosted with cell culture supplements and the antibiotic concentration in the growth media is reduced. The modified PEITMC-25 has suitable transfection efficiency with most proteins; however, there are other options available at moderate price that offer increased transfection efficiency and reduced cytotoxicity. Hype 5 (Oz Bioscience) increases yields compared to PEI or other reagents, at a considerably reduced cost compared to more expensive transfection reagents, such as 293fectin. The reagent type and DNA: reagent ratios can be optimized for individual expressions and the needs of the desired experiment."