Expression And Purification Of Hemolysin O And Human Retinol Binding Protein Of Streptococcus Pyogenes

Escherichia coli expression system which is a fully developed system remains the first choice in recombinant proteins expression. However, expression of foreign proteins in E. coli may result in inclusion bodies. In this study, two different foreign genes were cloned and expressed in different systems for investing solutions of inclusion bodies.Streptolysin O (SLO), an oxygen-sensitive protein contains thiol group, is characterized with cytolytic activity for many eukaryotic cells types, especially erythrocytes.it also causes pharyngitis, rheumatic fever and acute glomerulonephritis. In this work, the SLO gene from Streptococcus pyogenes was cloned into heat-shock vector pHsh, and was modified by removing initial 70 amino acids from N terminal. Low expression level and denatured proteins of recombinant SLO was observed in E. coli system, and additional analysis of optimum heat inducting condition showed no significant improvements. On the other hand, another clone was introduced into a cold-shock vector pEXC, expression level of recombinant SLO was significantly higher than that of pHsh, and there was no SLO observed in inclusion body form. Further optimization on different hosts (E. coli DH10B, JM109, BL21 and TOP10) showed that in E. coli BL21, the expression level of recombinant SLO reached the highest than other hosts. Additional point mutations on N terminal secondary structure based on software prediction were applied to remove the stem loop located between ribosome binding site (RBS) and initial translation site, but no obvious increase of expression was observed. At last,2 of 20 rare codons that were selected by a prediction of codon usage preference analysis were mutated synonymously, however, the expression level of recombinant SLO remained the same. Recombinant SLO were purified via two steps of affinity chromatography and cation exchange chromatography with a yield of 18 mg/L, western blot of purified SLO showed positive result against polyclonal antibody murine anti 6xHis, and the haemolytic activity assay showed that complete hemolysis occurred with a 80 ng/mL final concentration of purified SLO.Retinal binding protein (RBP) is a small molecular hydrophobic binding protein, which is considered to be an important indicator in tests of kidney and liver related diseases. In this work, four various expression vectors for E. coli (pHsh-rbp, pHsh-ex- rbp, pEXC-rbp and pTrc99a-rbp) and one for S. pombe (peno-GFAT-cpy-rbp) were constructed respectively, in order to study on expression levels of recombinant RBP in different systems. In E. coli expression system, expressions of recombinant RBP were accomplished by heat-shock, cold-shock, secretion and IPTG induction separately, in association with different optimizations of inducting conditions of different methods, however, SDS-PAGE results showed that no significant soluble expressions were observed, indicated that expression of RBP required modification systems that lack- in E. coli expression system. On the contract, S. pombe expression system has the similar mechanisms of protein secretion and post-translational modification of higher organism, recombinant RBP was successfully expressed in S. pombe system and in soluble forms, and recombinant protein were detected with anti-6xHis antibodies in western blot.