Recombinant Expression and Purification of T4 Phage Hoc, Soc, gp23, gp24 Proteins in Native Conformations with Stability Studies

Abstract

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli ). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

“…Phage proteins were used as bottom antigens in ELISA immunoassay. They were produced as optimized by Miernikiewicz et al [ 44 , 45 ]. Briefly, proteins were expressed in E. coli B834(DE3) F − ompT hsdS B (r B − m B − ) gal dcm met (DE3) (Novagen, Merck Millipore, Darmstadt, Germany) grown in LB high salt (10 g/L of NaCl) (Sigma-Aldrich, Poznań, Poland).…”

T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

“…Genes encoding short tail fiber ( 12 ) and its chaperone gp57 ( 57 ) were cloned to the expression plasmid pCDF-Duet1 (Streptomycin resistance; Novagen, USA), which contains two multiple cloning sites (MCS) under a T7lac promoter control. Amplification of gene 12 was conducted using polymerase chain reaction (PCR) and T4 total DNA ( Miernikiewicz et al, 2012 ) as a template. The primers were 12 forward 5′-AAAAGGATCCGATGAGTAATAATACATATCAACACG-3′ and 12 reverse 5′-AAAGCGGCCGCTCATTCTTTTACCTTAATTATG-3′.…”

“…Elutions were combined according to their purity and concentrated 2–5 times on Vivaspin centrifuge concentrators (Sartorius, Poland) at 18–20°C. Next, LPS was removed using EndoTrap Blue ( Miernikiewicz et al, 2012 ). The final preparation was dialyzed against PBS at room temperature and filtered through 0.22 μm PVDF filters.…”

Immunogenicity Studies of Proteins Forming the T4 Phage Head Surface

“…Phage proteins gp23*, gp24*, Hoc, and Soc were purified to immunological purity grade as described by Miernikiewicz et al (22). Briefly, genes coding for the proteins were cloned using Gateway technology into pDEST15 and pDEST24 vectors, allowing expression of recombinant products with glutathione S-transferase (GST) affinity tags, and expressed in the Escherichia coli expression system.…”