Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Abstract

ABSTRACT. The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS) 1 , L2: (GGGGS) 2 , and L3:(GGGGS) 3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins 19361Linker length affects HSA-fused ONC in P. pastoris ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 19360-19370 (2015) were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes

“…The GSA linker (GSAGSAAGSG) is rich in glycine and serine residues, and a similar linker, GSAGSAAGSGEF, has been employed to ensure correct folding of GFP and associated fusion proteins . Since both the NL and GSA linkers are flexible and Lst constructs containing these linkers presented almost the same structure, it appears that linker length may be crucial in Lst–linker–SiBP1 constructs, a phenomenon reported with other proteins. ,,− The difference in the length of the 16-residue NL linker and the 10-residue GSA linker was reflected in the observed differences in the size of the loop formed at the insertion point in the homology models (Figure ). The extra six amino acids in NL may provide additional space between Lst and SiBP1, making the LBD less affected by the rigidity of SiBP1.…”

Research progress of multi-enzyme complexes based on the design of scaffold protein

“…However, misfolding or inappropriate protein interactions may destabilize protein structures and affect activity levels (Hong et al 2006 ). Our previous investigation, we found that inapposite assembly owing to the spatial interference of the element domain may result in the loss of activity in the end-to-end fusion protein (Karp and Oker-Blom 1999 ; Hong et al 2006 ; Yang et al 2015 ). The construction of insertion fusion protein has a significant potential value in catalytic regulation and biosensors (Ataka and Pieribone 2002 ; Ribeiro et al 2019 ).…”

“…Factors such as flexibility, composition, conformation, length, and hydrophilicity of the amino acid should be considered when selecting a linker for the fusion proteins (Robinson and Sauer 1998 ; Arai R, 2001 ; Chen et al 2013b ; Fan et al 2015 ; Yang et al 2015 ; Li et al 2016 ; Huang et al 2021 ). According to the flexibility of the linker, they can be divided into flexible linkers and rigid linkers (Fig.…”

“…According to the study by G.G. Yang and coworkers, the length of linkers only affected the activity of rHSA-N-ONC (N = 0, 5, 10, and 15) but not its expression (Yang et al 2015 ). The short linkers have been successfully applied in many fusion proteins.…”

Biomolecular engineering for nanobio/bionanotechnology

“…The expression level of the fusion proteins had no relationship with the linker length. However, while the ONC moiety of the fusion protein without a linker (n = 0) showed no cytotoxicity toward tumor cells, this gradually improved with increasing linker length [338]. …”