Application Of Lectin From The Mushroom Laetiporus Sulphureus As Fusion Partner In Protein Expression And Purification

The development of genetic engineering technology,genomics and proteomics technology has promoted the progress of recombinant protein expression techniques.Recombinant proteins have been widely used in many fields of biological,medical,agricultural and animal husbandry and veterinary.However rapid and inexpensive purification of recombinant proteins is still a bottleneck for scale-up preparation of proteins.Affinity tags are highly efficient tools for protein purification.They enable one-step recovery of recombinant proteins from contaminating host proteins.By far,a variety of affinity tags have been used to facilitated expression and purification.The most widely used affinity tags include maltose-binding protein(MBP),glutathione S-transferase(GST),staphylococcal protein A(SPA),thioredoxin(TRX),small ubiquitin-like modifier(SUMO),and hexahistine(His).For using these tags,corresponding ligands(e.g.,transition metal ions for His-tag)have to be immobilized on a solid matrix.The binding of a tagged protein to its ligand is reversible,and the protein can be eluted under appropriate conditions.Concerning the cost of protein purification mainly associated with the cost of the stationary phased which immobilized with ligand,neither current available tags are not suitable for production of proteins in large scale,since most of commercial available purification matrix are expensive and only can be reused for several times.At present,there are a series of methods to solve these problems,such as changing culture conditions,the use of fusion tags.Fusion tag expression strategy is commonly used method,with the appropriate culture conditions,better expression and purification of recombinant proteins.On the one hand,the use of fusion tags to improve the soluble protein,on the other hand,select the appropriate fusion tag can achieve the purpose of purification of the recombinant protein.In this study,we developed a single-step purification method for LSL-tagged fusion proteins by using C-terminal deletion mutant of LSL as a fusion partner.Unmodified Sepharose-4B resin and lactose solution were used as a specific adsorbent and eluent,respectively.In order to remove the LSL-tag,tobacco etch virus(TEV)protease recognition sequence was placed downstream of LSL-tag in the expression vector,and LSL-tagged TEV protease,designated LSL-TEV,was also expressed in Escherichia coli.By using this method,LSL-tagged fusion proteins can be purified in one step with high yield in a cost-effective way,and native interested proteins can be easily produced in large scale.The thesis is composed of the following three aspects.1.Construction of expression vector with lectin-tag from mushroom Laetiporus sulphureusAccording to the sequence of the lectin gene of mushroom L.sulphureus(LSL),a truncated LSL(1-187aa)fragment with tobacco etch virus(TEV)protease recognition sequence was synthesized based on the codon usage of E.coli.LSL-tag expression vector was constructed by inserting the Nco I and Bam HI double digested gene into same sites of p ET28 a.The resulted plasmid was designated as p ET-LSL.The recombinant plasmids,p ET-LSL was introduced into BL21(DE3)bacteria host.The LSL was expressed under the control of T7 promoter as soluble forms in the cytosolic fraction of E.coli.The expressed LSL protein in the cleared cell lysate can be purified by one-step affinity chromatography on Sepharose 4B utilizing the intrinsic specificity of LSL for N-acetyllactosamine.2.Expression and purification of LSL-tagged model proteinsTo test the applicability of the LSL-tag expression vector,green fluorescence protein and the capsid(Cap)protein of the porcine circovirus type 2(PCV2)were chosen as model proteins and cloned into p ET-LSL expression vector.The resulted expression vectors,p ET-LSL-GFP and p ET-LSL-Cap were used to produce LSL-tagged fusion proteins.The results showed that both 90% of GFP and Cap proteins were expressed in soluble forms.The LSL-tagged GFP and Cap can be purified through one-step affinity chromatography on Sepharose 4B.The fact that the recombinant LSL-GFP exhibited specific fluorescence and the LSL-Cap can be recognized by anti-PCV2 polyclonal antibody demonstrated that the LSL tag won't affect the function of the model protein.3.Expression and purification of tobacco etch virus(TEV)proteaseTo facilitate the removal of LSL-tag from LSL-Cap fusion protein by using TEV protease,the codon-optimized TEV protease sequence was also inserted into p ET-LSL by using Kpn I and Bam HI restriction enzyme sites.The resulted plasmid p ET-LSL-TEV was introduced into BL21(DE3)host to express LSL-tagged TEV protease.The result turned out that 60% LSL-TEV was expressed in soluble form and can be purified through one-step affinity chromatography on Sepharose 4B.Importantly,the LSL-TEV protease exhibited good biological activities.After cleavage the LSL-tagged proteins by LSL-TEV protease and pass the digestion products through the Sepharose-4B column,the purified native interest proteins can be obtained easily.In conclusion,we have clearly demonstrated here that the lection LSL from the mushroom L.sulphureus can be used as a potent affinity tag that fulfils the following requirements:(i)the LSL-tag allows a one-step adsorption and purification of fused proteins;(ii)the LSL-tag enhances the solubility of target proteins and does not negatively affect the function of the target protein.It is worth pointing out that the Sepharose 4B column can be reused for many times after washing PBS containing 0.2 M lactose and then PBS.Moreover,the method developed in this study is much less expensive than conventional affinity purification methods,which require a ligand to be immobilized on a resin,because the Sepharose itself serves as both a resin and ligand for LSL-tag.