Expression And Purification Of Necl Family Proteins In The Extracellular Region Of Interaction With Intracellular Protein Screening

Nectin-like (Necl, also known as CADM or SynCAM) molecules are immunoglobulin(Ig)-like superfamily members. There are five members, Necl1, Necl2, Necl3, Necl4, and Necl5. Each of these includes a extracellular region composed by three Ig-like domains, a single transmembrane region and a short cytoplasmic region. Nectin-like molecules, like nectins, are Ca²⁺-independent cell-cell adhension molecules(CAMs). These proteins are specific or prominently expressed in the nervous system. Up to now, it is reproted that Necls play important roles in the formation of adhesion junctions, synapses and myelinated axons. Necl1 and Necl4 mediate axo-glial interactions, Schwann cell differentiation and myelination. Necl2 acts as a tumor suppressor and a regulator in synaptogenesis. The interactions among Necls which is mediated by the Ig-like domains and regulated by glycosylation, are important for their functions. In the previous studies of our lab, we resolved the crystal structure of the V domain of Necl1 and found that Phe82 was a key residue for the dimerization of Necl1 V domain. The other lab has reported the crystal structure of the first two Ig-like domains of Necl5 and its complexes with polioviruses.In order to study the structure of Necls, the insect cell expression system is chosen to express the extracellular regions of Necl1, Necl2, Necl3 and Necl4. The Bac-to-Bac^? Baculovirus expression system has been chosen to express these proteins. This is an efficient site-specific transposition system to generate Baculovirus for high-level expression of recombinant proteins. The coding sequences of the four genes are cloned from the cDNA of the adult mouse's brain, and the extracellular regions are inserted to the motified donar plasmids—the pFastBac^TMDual-Hemolin/Gp67-3C-His vectors. The donar plasmids are transformed to DH10Bac^TM E. coli, taken a site-specific transposition with the bacmid, and the exogenous gene fragments are inserted into the bacmid. Then the recombinant bacmid which contains the extracellular sequence of Necl proteins, are transfected to the insect cells Sf9 in order to get the recombinant baculovirus. Low MOI value of P1 generation of recombinant baculovirus are used to infect Sf9 cells, and amplify the virus required for expression. The method of real-time PCR is used to determine the titer of the virus.To determine the most appropriate conditions of the proteins, we carried out a series of tests. First, the recombinant virus with different signal peptides and different types of Necl1 (wild-type or glycosylation site mutant) are infected to insect cells, and the secretion and modification of exogenous proteins are detected. The results showed that there is no significant difference in the secretion between the two selected signal peptides, and there is no Necl1 protein glycosylation modification. Then, quantity of the exogenous protein expression using Sf9 or Sf21 cell lines is compared under the same conditions of infection.Third, two types of medium and six kinds of serum concentration are tried to optimize the culture conditions. Fourth, the Sf21 cells are infected with different MOI values. Fifth, collect the supernatant culture medium of the infected cells every 24 hours to detect the time point of the expression. Ultimately, the condition of Sf21 cells, Grace's insect medium, 2% fetal bovine serum, MOI of 8 and 3 days of culture time is chosen for the protein expression. And this condition is used to express these four kinds of proteins successfully. The Ni-column is used to purify the expressed proteins, different concentrations of imidazole are tried for elution, and at last 50mmol/L of imidazole is chosen as elution conditions, and get some high puritied extracellular region of Necl4 protein.Necls play a role with signal transduction pathways through intracellular domains. To further study the mechanism of protein Necl1, yeast two-hybrid system is adopted to screen the proteins which interact with the cytoplasmic tail of Necl1. The cytoplasmic tail sequence of human Necl1 gene was inserted into the "bait" vector pGBKT7 in frame. The recombinant pGBKT7-NECL1C was then transformed in to yeast cells and followed by screening of human fetal brain cDNA library. Nine different sequences by sequencing the positive clones(There are repeated clones) and five alternative proteins were obtained by the amino acids sequence blast. Using the GST pull down analysis, we confirmed that two of them can interact with the cytoplasmic tail of Necl1 in vitro.