In vivo cleavable fusion proteins increase protein yield while reducing unfolded protein response

Abstract

Purifying properly folded and functional recombinant proteins is crucial in various areas in research and the biopharmaceutical industry. Complexly folded proteins are of high scientific value and are often required in larger quantities. Due to complicated three-dimensional structures or numerous post-translational modifications, recombinant proteins often misfold, aggregate or are retained in the cell. An often-used technique to enhance protein folding and yield is attaching a protein tag to the target protein, which increases solubility and facilitates folding. To reduce subsequent adverse effects of the protein tag with function, structure or bioactivity of the target protein, in vitro removal of the protein tag by proteases is often needed. This process necessitates an additional purification step to remove the protease and protein tag from the target which will consequently decrease the final protein yield and can impact bioactivity and proper folding of the target. For these reasons, strategies without additional in vitro cleavage are beneficial but the number of established strategies to date is very limited. The present study depicts the endoprotease furin as a novel in vivo processing tool in human cells, cleaving recombinant proteins during secretion and in the trans Golgi network (TGN). The unique attribute of furin's activity in multiple compartments of the secretory pathway allowed for efficient in vivo processing of three model protein constructs: Interleukin-2 (IL-2), the receptor binding domain of severe acute respiratory syndrome coronavirus-2 BA1 (SARS-CoV2 Omicron), and an aggregation-prone firefly luciferase mutant of Photinus pyralis. Ultimately, this increased the yield of the mature and cleaved target protein secreted into the cell culture media. Utilizing endogenously cleaved IL-2 fusion protein led to enhanced levels of active IL-2 in the media, with a 1500 times stronger cellular response in HEK Blue™ IL-2 cells than commercially available IL-2 derived from Escherichia coli suggesting an increased activity due to IL-2’s native-like glycosylation patterns, when expressed in human cells. Cells expressing target protein without a protein tag demonstrated increased retention of the protein of interest. This observation uncovered a link between the unfolded protein response (UPR), the cell's protective mechanism, and the complexity and size of the target protein. Proteins with greater complexity and size show stronger expression of UPR specific markers such as binding immunoglobulin (BiP) and homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (HERPUD1) compared to smaller or less complex proteins. Moreover, high levels of protein yield using fusion proteins are linked to a beneficial impact of the fusion protein tags on the UPR in transient protein expression experiments. This study lays the foundation for producing in vivo cleavable fusion proteins in human cells and highlights a potential mechanism for improving cell productivity by cellular adaptation to ER stress.