Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag

Abstract

Whereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.

Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from Trapa bispinosa Roxb.

“…Escherichia coli was used as host bacteria for heterologous expression, producing inclusion bodies and inhibiting protein expression, but its activity remained unaffected. The low content of late catalytic might have been related to the particular processing mode of proteins in Escherichia coli , resulting in the protein not being wholly purified [ 47 , 48 ]. The specific reasons need to be investigated in further studies.…”

Computationally-Aided Modeling of Hsp70–Client Interactions: Past, Present, and Future

“…Limbo has also been used to identify or validate Hsp70 binding sites in many contexts, 95−99 including for rational design of binding sequences such as inhibitors. 100,101 ChaperISM 56 is a position-independent scoring matrix (PISM) predictor of Hsp70 binding to a particular sequence derived to reproduce quantitative binding data directly rather than distinguishing how peptides are classified as binders and nonbinders. On the basis of the same peptide binding data as in the Limbo study, 55 the source 10-mers were screened using FoldX 49 to computationally determine which 7-mers represent maximal affinity to DnaK in the original bound structure.…”

“…This result suggests that indeed the optimized sequence- and structure-based approach improved the model’s transferability across data sets. Limbo has also been used to identify or validate Hsp70 binding sites in many contexts, − including for rational design of binding sequences such as inhibitors. , …”

Coiled-Coil Based Inclusion Bodies and Their Potential Applications

“…Moreover, a subsequent study has demonstrated that the IBs formed by the fusion of ssTorA to ovalbumin (OVA)-derived epitopes can act as an antigenic vaccine formulation for T cell response (Schetters et al, 2020). Mutagenesis-screening of the ssTorA sequence has allowed the identification of improved versions of the tag with enhanced IB-formation properties (Jong et al, 2019).…”