Recombinant Escherichia coli Peptidyl-prolyl cis-trans isomerase C (ppiC)

Introduction

Recombinant Escherichia coli Peptidyl-prolyl cis-trans isomerase C (PpiC) is a cytoplasmic enzyme belonging to the parvulin family of folding catalysts. It accelerates protein folding by isomerizing peptidyl-prolyl bonds, a rate-limiting step in conformational changes. PpiC is encoded by the ppiC gene and is one of six cytoplasmic PPIases in E. coli, playing specialized roles in substrate recognition and stress response . Its recombinant form, often engineered with tags for purification, enables detailed biochemical and functional studies.

2.1. Domain Architecture

PpiC is a 17.8 kDa protein with a conserved parvulin-type fold. Key structural elements include:

• Active site: Histidine-81 (H81) and Methionine-57 (M57) are critical for catalytic activity and substrate binding, respectively .

• Substrate-binding pocket: A hydrophobic groove accommodates proline-containing peptides .

2.2. Enzymatic Activity

PpiC exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity with a catalytic efficiency ($k_{cat}/K_m$) of $1.69 \times 10^7 \, \text{M}^{-1}\text{s}^{-1}$ for the substrate succinyl-Ala-Leu-Pro-Phe-4-nitroanilide . This efficiency is comparable to other parvulins but lower than cyclophilins like PpiB, the major cytoplasmic PPIase in E. coli .

Table 1: Comparative PPIase Activity in E. coli

3.1. Identified Substrates

Pull-down assays and immunoprecipitation studies reveal PpiC interacts with:

• AhpC (alkyl hydroperoxide reductase): Requires PpiC’s PPIase activity for proper folding .

• GrxB (glutaredoxin): Binding depends on H81 catalytic residue .

• NusG (transcription antiterminator): Aggregates in Δ6 ppi strains .

3.2. Phenotypic Analysis of Knockout Strains

• Viability: Δ6 ppi strains (lacking all cytoplasmic PPIases) are viable only on minimal media at 30–37°C, highlighting PpiC’s essential role under nutrient-limited conditions .

• Temperature sensitivity: Growth ceases at 43°C or 23°C, indicating thermal stress dependency .

4.1. Cloning and Purification

Recombinant PpiC is typically expressed in E. coli BL21 (DE3) using plasmids like pET24b, followed by nickel-affinity chromatography due to C-terminal His-tags . A 3xFLAG epitope tag has also been used for immunoprecipitation studies .

4.2. Mutational Studies

• H81A mutant: Lacks PPIase activity, disrupting interactions with AhpC and GrxB .

• M57A mutant: Retains partial activity but fails to bind substrates like DeoC and RbsB .

5.1. Antioxidant Enzyme Regulation

Though primarily studied in E. coli, recombinant PPIases from other organisms (e.g., Porphyra yezoensis) enhance antioxidant enzyme activities (e.g., catalase, thioredoxin reductase) under oxidative stress . This suggests conserved mechanistic roles applicable to biotechnological stress-response engineering.

5.2. Protein Folding Studies

PpiC’s role in folding proline-rich proteins like AhpC provides insights into redox homeostasis and bacterial virulence .