| Title: | Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii |
| Author(s): | Dalvie NC; Naranjo CA; Rodriguez-Aponte SA; Johnston RS; Christopher Love J; |
| Address: | "Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. clove@mit.edu. The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. clove@mit.edu" |
| DOI: | 10.1186/s12934-022-01905-2 |
| ISSN/ISBN: | 1475-2859 (Electronic) 1475-2859 (Linking) |
| Abstract: | "BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide" |
| Keywords: | *covid-19 Humans Mating Factor Protein Sorting Signals Recombinant Proteins/genetics/metabolism SARS-CoV-2 Saccharomyces cerevisiae/metabolism Saccharomycetales Aggregation Pichia pastoris Product quality Protein engineering Signal peptide; |
| Notes: | "MedlineDalvie, Neil C Naranjo, Christopher A Rodriguez-Aponte, Sergio A Johnston, Ryan S Christopher Love, J eng INV-002740/Bill and Melinda Gates Foundation/ P30-CA14051/CA/NCI NIH HHS/ England 2022/09/06 Microb Cell Fact. 2022 Sep 5; 21(1):180. doi: 10.1186/s12934-022-01905-2" |
