Construction Expression And Purification Research Of Citrus Huanglongbing Serralysin Protein

Citrus Huanglongbing or Citrus Greening Disease is the most destructive disease in the world Citrus production. It is the major cause of citrus decline. Efforts should be done to cope with this threaten to the world crops production. Until now, little was known about the mechanism how the bacteria affect Citrus plants. When searching the mechanism of how bacteria affect its host, gene and protein functions are concerned. And secreted proteins are one kind of these molecules. The relatively simple type 1 secretion system in Gram-negative bacteria is nevertheless capable of transporting polypeptides across the cell envelope. Its mechanism has been fully illustrated, it is the major way bacteria interacted with the host.In this study, we designed primers to clone COG2931 gene from Candidatus Liberibacter asiaticus genome (NC₀12985), then constructed a expression vector to produce recombinant protein, and induced high expressing of that protein. At last we purified the protein in a two steps way. These are our results:1. Extracted genome DNA from periwinkle leaves, and used PCR method and scanning electron microscope to confirm. Cloned Serralysin gene, gained a fragment of 2089bp. Sequencing and sequence alignment showed that, the fragment is a member of serralysin-like family, there is three conserved domains in its sequence, and there are three highly repeated domains in C terminal.2. We constructed a expression vector designed basing on pET-22b(+) vector in which Serralysin gene can co-expressed with His-Tag. Using this expression vector we produced constructed Serralysin protein. By means of inducing in different conditions, we found optimal effectors for its expression, that are E.coli cells grown in LB broth at 37℃with shake until OD600 reached to 0.6, induced in 0.4mM IPTG for 3.5 hours in 280℃.3. Two steps purification by Ammonium Sulfate Precipitation and Ni2+-NTA affinity chromatography were developed to yield serralysin protein. After purification, the protein is 99% in purity, and the total recovery percentage is 1%.