ABSTRACT
Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b 5 (CYT b 5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb 5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb 5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS-PAGE and western blot analysis.
ABSTRACT
The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the "alanine" alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔGtotal = -140.62 kcal mol-1), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔGtotal = -124.24 kcal mol-1). Here we report the dynamic behavior of change of "alanine" in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.
