| Purpose: To construct a new prokaryotic heterologous expression system of cellulose-binding domain,pentamer domain and ScFv(single chain fragment variance)fusion proteins,and check/test its expression in E.coli,and purification via affinity chromatography based on Ni-NTA and cellulose tandem purification process.Method: 1.Vector construction.In this work,as an example,the ScFv derived from the monoclonal antibody against soy Bowman-Birk protease inhibitor(anti-BBI)was used to test the new prokaryotic expression and affinity purification system.The anti-BBI ScFv was amplified from synthesized oligonucleotide fragments overlapping with one another,and cloned into various expression vectors/constructs with or without the cellulose-binding domain and/or pantamerization domain,i.e.1)pET22b-anti-BBI only,2)pET22b-anti-BBI-pantamerization domain,3)pET37b-(cellulose-binding domain)-anti-BBI only;,and 4)p ET37b-(cellulose-binding domain)-anti-BBI-pentamerization domain.All the vectors contain a poly-His tag at the C-terminus.2.Protein expression.The various prokaryotic expression vectors obtained above were introduced into E.coli strain BL21.The heterologous proteins were induced with0.5 m M IPTG at 18?,and after induction for overnight cells were harvested and lysed by sonication.Cell lysate was isolated,and the expression of proteins was examined by SDS-PAGE.Insoluble proteins in inclusion bodies were solublized by 8M urea-containing buffer.3.Protein purification.The cell lysate or inclusion body solution was incubated with the pre-treated Ni-NTA resin,and after washing with an appropriate buffer the fusion proteins were eluted with 0.4 mM imidozol.The elutes were examined by SDS-PAGE for purification efficiency.4.Protein renaturation.The purified denatured proteins were dialysed with buffers of decreasing urea concentrations gradually,and the renatured proteins were cleared by centrifugation.5.Cellulose affinity chromatography.The renatured proteins were incubated with crystalline cellulose,and the contaminants were washed away by repeated washing.The proteins bound to cellulose were eluted and checked by SDS-PAGE,which was stainedwith Commassie Blue,for comparison of affinity to cellulose.Results:1.Multiple prokaryotic expression vectors were constructed with anti-BBI ScFv alone,or fused with cellulose-binding domain,and/or pentamerization domain,including 1)pET22b-anti-BBI ScFv only;2)pET22b-anti-BBI-pentamerization domain fusion protein;3)pET37b-cellulose-binding domain-anti-BBI fusion protein;4)pET37b-cellulose-binding domain-anti-BBI-pentamerization domain.2.The E.coli strain BL21 was used as the expression host of the above multiple constructs.The bacteria was induced to express the target proteins via IPTG at a relatively high level.Most of the proteins were located in inclusion body,and only a minor fraction was in the cell lysate.3.The fusion proteins were purified via Ni-NTA resin,which bound to the poly-His tag located at the C-termina of the target proteins with relatively high content of contaminants.4.The purified and renatured proteins retained the activity of binding to cellulose,and the proteins after cellulose affinity chromatography showed higher purities.5.A further pentamerization process increased the cellulose-binding affinity by at least two times,which can be used for a higher purity.Conclusion:1.Cellulose-binding domain,pentamerization domain,ScFv fusion proteins can be expressed in E.coli at a relatively high level.2.The solubility of Sc Fv(single-chain fragment variance)and its varied fusion forms are similar,which may be due to the nature of Sc Fv,and a denature-purification process is needed for protein purification.After renaturation the activity of the proteins can be recovered.2.Cellulose is an appropriated matrix for purification of proteins with cellulose-binding domain(CBD).3.Introduction of a pentamerization domain increased the cellulose-binding affinity,and is an ideal alternative for high purity protein purificaion. |
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