Expression, Purification And Characteristics Of A Truncated Human Delta-like1, A Ligand Of Notch Receptors

The Notch signaling pathway plays a pivotal role in proliferation,apoptosis,and so on in both embryonic and postnatal development,and is a potential therapeutic target for human diseases such as cancer.Notch is a family of typeâ… transmembrane proteins that includes four members in mouse and man(Notch1-4).Five Notch ligands have been identified in mammals, including Jaggedl,2,and Delta-like1,3,4.Notch ligands are also typeâ… transmembrane proteins,with sophisticated structural motifs including an N-terminal DSL motif that is essential for Notch-binding,and varying number of EGF like repeats.The human Delta-like1(hDll1)is a homolog of the Dro-sophila.Delta,and is composed of 723 amino acids,with one DSL and eight EGF-like repeats.These structural properties,as well as the complicated intracellular trafficking pathway during synthesis,have made it difficult to manufacture Notch receptors or ligands in either eukaryotic cells or Escherichia coli,although production of these molecules has been greatly desired due to their therapeutic potentialsThe thesis includes two parts as follows:1.Expression and fermentation of a soluble human Delta-like1 protein in Escherichia coilTo express hDll1 in Escherichia coli,we amplified the N-terminal sequence of hDll1 from a human cDNA library by PCR,and constructed the GST-hDll1 gene, which was successfully expressed in Escherichia coli.The effects of culture media, carbon source,culture temperature,pH,and dissolved oxygen,on the growth of Escherichia coli and the expression of GST-hDll1 protein were systematically investigated.Firstly,the M9 medium was selected from several kinds of basic culture media,and was adapted for the growth of bacteria and the expression of GST-hDll1. By measuring bacterial growth with OD₆₀₀and protein expression with SDS-PAGE, the optimum conditions were established as following:temperature,30℃;initial pH7.2;the volume of culture broth in shaking flask,50mL;approximate rotation speed,210 r/min;carbon source,glycerol.Induction was achieved by addition of IPTG(0.6 mmol/L)when the OD₆₀₀of the bacteria reached 0.7-1.0,and the induction was permitted for 5h at 30℃when the OD₆₀₀of bacteria and expression of GST-hDll1 were 3.36 and 42.9%,respectively.Based on the results of shaking flask cultivation,we compared the batch culture with fed-bateh culture,and found that the fed-batch culture was much better adapted to the growth of bacteria and the expression of GST-hDll1 than the batch culture.2.Recovery and renaturation with simultaneous purification of soluble human Delta-like1 inclusion bodies by LCThe harvested bacterial paste was resuspended in buffer,and lysed by sonication,followed by centrifugation.After washing,the inclusion bodies were dissolved,refolded and purified simultaneously using protein folding liquid chromatography(AFC,SEC,HPHIC and IEC).(1)The purified GST-hDll1 was more than 97%pure with a mass recovery of 89%using AFC;(2)The purified GST-hDll1 was 95%pure using urea gradient SEC,with mass recovery of 62%.The eluted GST-hDll1 protein using urea gradient showed a single peak after RP-HPLC and the purified product had a purity of more than 99%;(3)The chromatographical conditions should be optimized further to get better purification results using HPHIC and IEC for refolding and simultaneous purification of GST-hDll1;(4)The resulting aim protein had a molecular weight of 39 kD when the result was confirmed with SDS-PAGE and MALDI-TOF.Compared with GST,the purified GST-hDll1 had an activity of triggering Notch receptors,as assessed using a reporter assay.