Constructing Prokaryotic Vector Of LSECtin-CRD Gene,Expression And Purification Of Its Recombinant Protein

Abstract Objective:Liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin) is a type Ⅱ integral membrane protein and plays important roles in the innate and adaptive immune responses, either as pathogen recognition receptors or cell adhesion receptors. LSECtin contains so-called carbohy-drate recognition domains (CRDs) that bind carbohydrate structures in a calcium (Ca2+)-dependent manner. This study was carried out a series of LSECtin-CRD vector construction and expression of recombinant proteins in Escherichia coli, in order to obtain a sufficient amount of soluble recombinant protein of LSECtin-CRD and provide theoretical basis for study on the information of the mobility and structure changes of LSECtin-CRD by the application of site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR). Methods:(1) Construction of expression vector:Planed to construct three different expression vectors,[PET-22b(+)-LSECtin-CRD、 PGEX-6p-1-LSECtin-CRD and PGEX-6p-1-LSECtin] Primers of DNA fragments (LSECtin and LSECtin-CRD) were designed and synthesized according to the purpose of the experiment, products of PCR amplification were identified by agarose, after identification gel recycling were done, double digestion of PCR fragments were identified to be correct, then obtain purified small fragments of exogenous DNA by gel recycling. After that, DNA fragments were cloned into the prokaryotic expressing vector, and transformed into the host of competent cell DH-5a for enlargement culture, The expression vectors [PET-22b(+) and PGEX-6p-1] were isolated and purified, and verified by double digest. DNA sequencing was performed by invitrogen Company.(2) Induced expression of recombinant protein:three expression vectors were identified to be correct, and transformed into two different host bacteria [BL21(DE3) and Origami (DE3)] for further higher expression, Selected monoclonal and expanded cultivation were identified to be correct by double digestion, then recombinant proteins were expressed and identified by SDS-PAGE and Western blot.(3) Purification and renaturation of recombinant protein:Choosed better strains of expression recombinant protein [PGEX-6p-1-LSECtin-CRD], collected the bacterial protein, renatured and purified. The purified Protein was examined by SDS-PAGE. Results:Three different expressing vectors (pET-22b(+)-LSECtin-CRD,pGEX-6P-1-LSECtin-CRD, PGEX-6P-1-LSECtin) were constructed and expressed. Large amounts of recombinant protein was successfully expressed in two different host bacteria [BL21(DE3) and Origami (DE3)], but they were in the form of inclusion body. Choosed finer expression recombinant bateria [Origami (DE3)-PGEX-6p-1-LSECtin-CRD], through the expression, purification and renaturation, gained plenty of soluble protein (PGEX-6P-1-LSECtin-CRD). Conclusion:Three different expression vector [PET-22b(+)-LSECtin-CRD, PGEX-6p-1-LSECtin-CRD, PGEX-6p-1-LSECtin] were successfully constructed, all of them could express large amounts of recombinant proteins in Escherichia coli, but the total as inclusion body, through the purification and renaturation, getting enough amounts of soluble protein.