Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.
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.
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
Paulina
P
Miernikiewicz
Barbara
B
Owczarek
Agnieszka
A
Piotrowicz
Barbara
B
Boczkowska
Kamila
K
Rzewucka
Grzegorz
G
Figura
Andrey
A
Letarov
Eugene
E
Kulikov
Agnieszka
A
Kopciuch
Kinga
K
Switała-Jeleń
Anna
A
Oślizło
Katarzyna
K
Hodyra
Jerzy
J
Gubernator
Krystyna
K
Dąbrowska
D017122:Bacteriophage T4 / Q000235:genetics*; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008070:Lipopolysaccharides / Q000302:isolation & purification; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D036022:Capsid Proteins; C108525:HOC protein, T4 phage; D008070:Lipopolysaccharides; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C074187:gene 24 protein, Enterobacteria phage T4; C064517:gp23 protein, Bacteriophage T4; C103392:small outer capsid protein, bacteriophage T4