Small Ubiquitin-like Modifier Mediated Purification Technique And Its Application

Nowadays, the demands for highly purity active proteins in areas of medicine, biocatalysis andemerging technologies are increasing rapidly. But in industry, large-scale recovery and purifcationof these proteins are both difficult and expensive. Recent studies have found that affinity tags arehighly efficient tools for protein purification. They allow the purification of almost every proteinwithout any prior knowledge of their biochemical properties. Currently, the study of novel affinitypurification tags and tag removal methods is an important research direction of affinity purificationtechnique. In allusion to the hot and difficult problems in the purification of recombinant proteins,we have carried out our study on the combined use between the small ubiquitin-like modifier andnon-chromatography purification tag-elastin like polypeptides (ELPs) and chromatographypurification tag-ribosomal protein L2，and finally a novel affinity purification technique wasdeveloped in this paper. The main research contents and results are as follows:1. Monomer ELP genes of ELP[KV7F-9] and ELP[EV7F-9] were oligomerized by recursivedirectional ligation to create genes for ELPs with different molecular weights and express thecorresponding fusion proteins. The ELPs and its fusions were purified by inverse transition cycling.The inverse transition temperature was measured at different buffer pH. The results demonstratedthat the inverse transition temperature of cationic or anionic ELPs was depended on the buffer pH,as the buffer pH got closer to the pI value of ELPs, the inverse transition temperature of ELPsturned lower. Effect of ELPs length on the expression and purification of fusion proteins wassignificantly. Reducing the size of the ELP tag increases the expression yield of fusion proteins, butdecreases the purification recovery of fusion proteins. The position of the fusion protein also has asignificant impact on the expression and purification of ELP fusion proteins. Placing the targetprotein (EGFP and Trx) at the N-terminus of the ELP (protein-ELP) results in a higher expressionlevel, which also translates to a greater yield of purified protein except EGFP in yield. Smallubiquitin-like modifier (SUMO) fusion proteins have an increase in expression levels and yieldscompared with their unfused counterparts. However, the inverse phase transition of the ELP-SUMOfusion tag was difficult to be trigerred by the addition of NaCl. Thus, the hot spin efficiency ofELP-SUMO fusion tag became low.2. According to the adsorption mechanism of ribosomal protein L2and its truncated mutantson silica particles, we have selected several materials, and then a relatively good Si-containgmeaterial (diatomite filter aid STD) was chosen as the affinity purification media for ribosomalprotein L2. This paper have studied the difference of the express level and purification property ofL2(1-273) and the L2(1-60,203-273), and finally confirmed that the L2(1-60,203-273) was used aspurification tag instead of the L2(1-273), and the diatomite filter aid STD was used as affinityadsorption in the purification of fusion proteins. 3. The L2(203-273) has higher pI and proportion of basic amino acid. After the comparativestudy of the different adsorption properties of the two deleted proteins of L2on diatomite filter aidSTD, we could know that the L2(203–273) was the sufficient region for the adsorption of ribosomalprotein L2on diatomite. Then the adsorption properties of L2(252-273) and L2(237-273) ondifferent adsorbents were also studied. Our studies demonstrated that the L2(203-273) orL2(252-273) had larger adsorption capacity and quicker adsorption rate, which also demonstratedthat the surface probability of basic amino acids in polypeptides is the deciding factor on theelectrostatic interaction between basic polypeptides and adsorbents. Buffer pH also have asignificant effect on the adsorption of L2(252-273) on diatomite and carboxymethyl chitosanmagnetic nanoparticles. The adsorbent with higher pI was less easy to be influenced by pH,compare with the adsorbents with lower pI. The study of adsorption kinetics and isotherm foundthat the L2(252-273) fusion proteins could adsorbed onto the diatomite of which the pI is lower, andget a faster adsorption rate. But the adsorption capacity of the L2(252-273) onto the carboxymethylchitosan magnetic nanoparticles was higher than diatomite. When the L2(252-273)-SUMO fusionprotein technology was used to purify the EGFP, we could get purified EGFP of which purity andpurification recovery all beyond90%.4. As the cation exchange resin has better mechanical stability, L2(252-273)-SUMO fusionprotein purification method can use adsorption, cleaning and enzyme cleavage purification protocolwhich has the advantages of simple and time saving over the purification protocol with adsorption,cleaning, elution, enzyme cleavage and adsorption. L2(252-273)-SUMO fusion protein purificationmethod can be effectively applied to purify the recombinant catalase (KatA) using cation exchangeresin as the affinity matrix. This purification procedure was used for purifying the KatA, andobtained a purification fold of30.5, a high specific activity of48,227.2U/mg and a high activityrecovery of74.5%. This catalase purification recovery was higher than most of the other catalasepurification methods. The biochemical properties of this purified catalase agreed well with theprevious reports about Bacillus subtilis catalase. But due to the limitation of E. coli endogenousheme pool, the specific acitivity of recombinant catalase was lower.5. As the SUMO protease has several rare codon clusters, using rare codon enhancedEscherichia coli-Escherichia coli Rosetta (DE3) as the express host can significantly increase theexpression of SUMO protease in E. coli. The poly lysine tagged SUMO protease had higher robustSUMO cleavage efficiency than that of L2(1-60,203-273) tagged SUMO protease at the sameprotein concentration, but had simillar SUMO cleavage efficiency with equal molar concentration.This result indicates that L2(1-60,203-273) and poly lysine fusion tag has the similar effect on thespecific acitivity of SUMO protease. Both cation exchange resin and magnetite nanoparticles can beused to purify the poly lysine tagged SUMO protease. The maximum static adsorption capacity ofUlp1-K10on Amberlite Cobalamion and magnetite nanoparticles reached36.8and211.4mg/g,respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion.