Comparison of the Effect of Two Purification Methods on the Immunogenicity of Recombinant Outer Membrane Protein H of Pasteurella multocida Serovar A:1.

Arunee Thanasarasakulpong,Thanya Varinrak,Weerapongse Tangjitjaroen,Dirk Pfeiffer,Nattawooti Sthitmatee,Takuo Sawada,Pichayanut Poolperm

doi:10.1155/2016/2579345

Abstract

Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinity chromatography but this adversely affects its immunogenicity. The current study presents the results from an intervention study comparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography and native OmpH purified using electroelution and a nonimmunized control group. Chickens immunized with rOmpH purified using electroelution produced the highest ELISA antibody levels against P. multocida strains. Chickens in each of the 5 treatment groups were split into two subgroups for challenge with two different P. multocida strains. The average number of adhesions to CEF cells was statistically significantly lower in sera from chickens immunized with rOmpH or native OmpH purified using electroelution than in those of the three other treatment groups. The survival amongst chickens immunized with rOmpH or native OmpH purified using electroelution indicated high levels of protection. In contrast, survival probability was zero or low in the groups immunized with rOmpH purified using affinity chromatography and in the nonimmunized group. These findings show that the rOmpH purified using electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P. multocida infection.

Highlights

Recombinant proteins with 6×His-tagged protein are routinely purified by a Ni-NTA affinity chromatography as recommended by their manufacturers
Luo et al [1] and Rimler [2] suggested that this process resulted in a change in the structure of the recombinant outer membrane protein H of avian Pasteurella multocida strain X73 affecting its immunogenicity
Sthitmatee et al [3] successfully improved the immunogenicity of recombinant outer membrane protein H (rOmpH) using a hybrid condition of affinity chromatography to purify rOmpH

Summary

Introduction

Recombinant proteins with 6×His-tagged protein are routinely purified by a Ni-NTA affinity chromatography as recommended by their manufacturers. Sthitmatee et al [3] successfully improved the immunogenicity of rOmpH using a hybrid condition of affinity chromatography to purify rOmpH This method is unstable, is of low reproducibility, and results in a high loss of protein yield [3]. This suggests that affinity chromatography may be unsuitable for purification of this recombinant protein. The method employs polyacrylamide gel electrophoresis (PAGE) which is easy to perform and has high resolution and good reproducibility This system has advantages in terms of having high loading capacity of sample protein and allowing easy monitoring of the elution process. The method has been used for purification of a native form of OmpH

Methods

Results

Conclusion