Modifications in the Kex2 P1’ cleavage site in the α-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris

Aggarwal, Sudhir; Mishra, Saroj

doi:10.1007/s11274-021-03167-3

Cited by 8 publications

(4 citation statements)

References 38 publications

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“…It was found that the Ans in the P1’ site of Kex2 could increase over fourfold of luciferase as much compared to the Cys in the P1’ site [ 35 ]. Substitution of Glu by Val/Ala at Kex2 cleavage site P1’ could increase by 3 times the production of extracellular colony−stimulating factor [ 43 ]. However, there were no studies on P1’ site optimization to improve the yields of AMPs so far as we know.…”

Section: Discussionmentioning

confidence: 99%

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

et al. 2023

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Pichia pastoris is the widely used expression system for producing recombinant secretory proteins. It is known that Kex2 protease plays a vital role in the process of protein secretion, in which the P1’ site affects its cleavage efficiency. To enhance the expression level of fungal defensin-derived peptide NZ2114, this work attempts to optimize the P1’ site of Kex2 by replacing it with 20 amino acids in turn. The results showed that when the amino acid of the P1’ site was changed to Phe (F), the yield of target peptide significantly increased from 2.39 g/L to 4.81 g/L. Additionally, the novel peptide F-NZ2114 (short for FNZ) showed strong antimicrobial activity against Gram-positive (G+) bacteria, especially for Staphylococcus aureus and Streptococcus agalactiae (MIC: 4–8 μg/mL). The FNZ was very stable and retained high activity in various conditions; in addition, a low cytotoxicity and no hemolysis were observed even at a high concentration of 128 μg/mL, and a longer postantibiotic effect was reached. The above results indicate that this engineering strategy provided a feasible optimization scheme for enhancing the expression level and druggability of this antimicrobial peptide from fungal defensin and other similar targets by this updated recombinant yeast.

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Section: Discussionmentioning

confidence: 99%

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

et al. 2023

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“…We also showed the insertion of the αSP EAEA residues can also eliminate these N-terminal variants. Others have demonstrated that mutation of the + 1 residue of the KEX2 cleavage site may impact signal peptide processing and improve the secreted titer of recombinant proteins [ 38 ]. In most studies that evaluate the impact of the EAEA residues, however, the residues remain on the recombinant protein; that is, cleavage of the EAEA residues by STE13 does not occur [ 20 , 21 ].…”

Section: Discussionmentioning

confidence: 99%

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

et al. 2022

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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.

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“…We also showed the insertion of the αSP EAEA residues can also eliminate these N-terminal variants. Others have demonstrated that mutation of the +1 residue of the KEX2 cleavage site may impact signal peptide processing and improve the secreted titer of recombinant proteins [34]. In most studies that evaluate the impact of the EAEA residues, however, the residues remain on the recombinant protein; that is, cleavage of the EAEA residues by STE13 does not occur [19,20].…”

Section: Discussionmentioning

confidence: 99%

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

Dalvie

Naranjo

Rodriguez-Aponte

et al. 2022

Preprint

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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 will enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.

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Modifications in the Kex2 P1’ cleavage site in the α-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris

Cited by 8 publications

References 38 publications

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

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