The Expression And Purification Of Protein ClACP And ClBirA From Candidatus Liberibacter Asiaticus

Citrus huanglongbing (HLB) is a worldwide spread destructive disease of citrus. At present, the main prevention methods involve excavating diseased trees timely, controlling the reproduction and diffusion by propagation media of citrus psylla, strengthening quarantine inspection of seedlings, as well as cultivating strong trees and so forth. Whereas antibacterial agents that can effectively inhibit pathogenic bacteria from citrus huanglongbing have not yet been achieved.In this research we primarily use molecular biology to investigation to the structures of three related proteins in two different pathways of Candidatus Liberibacter asiaticus: ?-hydroxyacyl-ACP dehydratase(ClFabZ) and acyl carrier protein(ClACP) in fatty acid synthesis, ClBirA produced during synthesis of biotin. The knowledge of interaction relationship between proteins ClFabZ and ClACP on molecular level and the structure of protein CIBirA can provide insight into screening antibacterial agents.It is vital to understand the interaction relationship between ClFabZ and ClACP since it can locate the interaction sites and active sites in protein ClFabZ. Therefore, we first acquired the two aforementioned proteins with relatively high purity after a series of procedures including genetic cloning, protein expression and purification. And we preliminarily identified such interaction through molecular sieve co-purification. Then we conducted massive screening and optimization for crystallization condition of protein complex. But, unfortunately, we haven't obtained protein complexes crystals for solving structure.Biotin protein ligase plays key roles in synthesis of biotin, and shares two distinct biological functions with different ways. Protein purification and crystallization of ClBirA confer high significance for structural and functional research on protein ClBirA. During the processes of cloning, expression and purification, we confirmed that CIBirA expressed best in carrier pET28a-sumo after many attempts and gropes including constructions of three different expression vectors and verifying suitable conditions for separation and purification. Furthermore, using Ni2+column affinity chromatography and Q column separation and purification, we obtained CIBirA protein with good solubility, high stability, high purity and uniform states. In summary, this study laid a foundation for further investigation on screening of CIBirA protein crystallization conditions.