Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

Abstract

At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75 C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90 C. MutC has retained more than 50% activity even after incubation at 100 C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.

In silico Approach to Elucidate Factors Associated with GH1 β-Glucosidase Thermostability

“…Decrease of polar amino acids in thermostable enzymes contributes to thermostability by minimizing cavities, Gln-and Asn-induced deamidation, and Cys, Ser and Thr oxidation at higher temperatures. This finding is in agreement with previous reports 13,125,126,129 . In contrary, charged amino acids (Glu, Asp, Lys, Arg) are higher in HT-BGLs and T-BGLs than M-BGLs (P< 0.05, Table 5).…”

“…Pro has been used to increase protein thermo-stability and can be considered, here, a potential hotspot to enhance thermostability of BGLs 124 . Similarly, Met, Asn, and Ser are thermo-labile that undergo either oxidation or deamination (Asn) at elevated temperature and are therefore less common in the thermostable protein 125,126 . Indeed, the substitution of Ser by Ala in thermophilic protein is widely reported 100 .…”

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

“…Other groups have tackled deamidation a step further back, using re-cloning and protein engineering strategies. Labile Asn and Gln residues can be removed from the product altogether as reported for two enzymes, 59,60 providing that there is no impact on product function or structure. However in many cases, this is either not possible or creates a virtually insurmountable problem from a regulatory and product licensing point of view.…”

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…The stressed studies conducted to assess the CQAs for a molecule may identify targets for improvements, based on information about the susceptibility of certain amino acid modifications. Protein engineering has also been employed to reduce deamidation and isomerization in lipases and mAbs with well‐characterized structural information by stabilizing secondary structures around the susceptible sites (Bhanuramanand, Ahmad, & Rao, ; Patel et al, ; Yang, Zhao, et al, ). Overall, cell line development has proven effective at controlling endogenous PTMs with known mechanism but may have limited capacity in resolving other extracellular PTMs without detailed structural information.…”