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1. Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Author

M. Moradi Barzadd, Anna-Luisa Volk, Diane Hatton, Raymond Field, Aman Mebrahtu, Paul Varley, R. G. Roth, Veronique Chotteau, N. Wistabacka, Fredrik Edfors, Chih-Chung Kuo, Magdalena Malm, Nathan E. Lewis, Ronia Razavi, T. Graslund, Johan Rockberg, David Kotol, and M. Lunqvist

Subjects
Secretory Pathway, HEK 293 cells, Bioengineering, PDIA3, CHO Cells, Biology, Applied Microbiology and Biotechnology, Recombinant Proteins, Cell biology, law.invention, Transcriptome, Cricetulus, HEK293 Cells, law, Cricetinae, Recombinant DNA, Animals, Humans, Secretion, HSPA8, Gene, Secretory pathway, Biotechnology
Abstract

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Published
2022

2. Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of affibody molecules site-specifically labeled with (99m)Tc, (111)In, and (125)I.

Author

Hofstrom C, Orlova A, Altai M, Wangsell F, Graslund T, and Tolmachev V

Subjects
Animals, Cell Line, Cell Line, Tumor, Cysteine chemistry, Cysteine metabolism, Female, Glutamic Acid chemistry, Glutamic Acid metabolism, Histidine chemistry, Histidine metabolism, Liver metabolism, Mice, Oligopeptides chemistry, Radioisotopes, Receptor, ErbB-2 metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins pharmacokinetics, Tissue Distribution, Indium Radioisotopes, Iron Radioisotopes, Liver drug effects, Organotechnetium Compounds, Recombinant Fusion Proteins chemical synthesis, Recombinant Fusion Proteins isolation & purification
Abstract

Affibody molecules are a class of small (∼7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [(99m)Tc(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with (111)In, (99m)Tc, and (125)I at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.

Published
2011
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