The Experimental Study On Construction And Application Of A Novel Secretion Expression System PQABPS/E.Coli M15 Utilizing RsaA Secretion Apparatus Of Caulobacter Crescentus

Obtaining soluble proteins often constitutes a bottleneck in the production of recombinant proteins for drug developments, structural studies and proteomics. This can be accounted for, at least in part, the lack of efficient secretion systems capable of large-scale exportation of recombinant proteins. Secretory expression of recombinant proteins in Escherichia coli has exhibited several advantages over intracellular production, including: 1) to facilitate the downstream process; 2) to enhance the biological activity; 3) to increase the stability and solubility of recombinant proteins. Among the five major secretion systems (type I, II, III, IV, and V) that can export protein into the extracellular space. Type I (ATP-binding cassette) secretion system featuring a number of striking features has been recognized to be an attractive candidate for development of extracellular expression systems of recombinant proteins.In E. coli, the a-haemolysin (HlyA) transporter is a representative member of the type I transporter family. Indeed, it has been utilized extensively. Despite many advantages, the HlyA secretion pathway has a number of drawbacks. Probably, there is not sufficient room for further improvement of HlyA-based secretion systems, demanding the exploration of more efficient and less restrictive secretion systems for recombinant protein exportation.RsaA (regular surface array protein) secretion system in Caulobacter crescentus, which mediates the secretion of surface layer protein (SLP) is considered as a promising candidate for development of improved recombinant protein secretory delivery systems. Theoretically, as long as the four secretion element genes rsaD, rsaE, rsaFa and rsaFb were cloned and expressed, the heterologous proteins fused to RsaAs, the rsaA C-terminal secretory signal, will be secreted to the extracellular medium. However, the regulatory elements of C. crescentus couldn't be recognized by the host E. coli. So some other regulatory elements that can work in E. coli should be involved.In this work, we cloned a series of genes encoding exporter proteins rsaD, rsaE, rsaFa, rsaFb, the C-terminal signal sequence rsaAs and three other exogenous regulatory components respectively. These genes and the foreign regulatory components were directionally inserted into pQE30 vector, resulting in the plasmid, termed as pQABPS. Then four heterologous proteins (GFP, EspA UreB and hIL24) from different origins and of different sizes were fused to the C-terminal signal for evaluation of the extracellular secretion capacity of the newborn plasmid pQABPS.The study has been involved in the following aspects:1. Selection and determination of exogenous regulatory componentsSix gene expression regulatory components—strong terminator rrnBT1, strong constitutive T5 promotor PTS and its mutant PTW, moderate constitutiveβ-lactamase promoter PB and PBS (its SD sequence was included), Epsilon (UUAACUUUA, T7 phage gene 10 translation enhancer), along with Shine-Dalgarno (SD) consensus sequence AAGGAG (abbreviated as ESD), were cloned. SDS-PAGE analysis showed that the reporter protein GFP was constitutively expressed under the regulation of them. Two optimized group rrnBT1-PTS-ESD and rrnBT1-PB-ESD were selected to regulate the expression of four secretion element genes.2. Development and evaluation of the extracellular secretion vector pQABPS1) Four Caulobacter crescentus RsaA secretion elements rsaD, rsaE, rsaFa and rsaFb and three exogenous regulatory components were directionally inserted into pQE30 vector, generating the interested plasmid designated pQABPS.2) The reporter protein GFP were subjected to expression assays using this newly-developed expression system, pQABPS/E. coli M15. The hybrid protein in the culture supernatant was detected by SDS-PAGE and Western bloting. The secretion amount was about 1.0mg/L. To test whether the target protein was secreted through the special RsaA transporter, expression of four structural proteins RsaD, RsaE, RsaFa and RsaFb was tested by RT-PCR and SDS-PAGE. As a result, the GFP hybrid protein was exported from the E. coli cells in an RsaA secretion system dependent manner, but not a nonspecific leakage after long-time cultivation.3)The stability of the pABPS plasmid in E. coli M15 was analysed. It showed that the plasmid had good genetic stability in M15 with selective pressure.3. Application and optimization of the pQABPS vector1) Three other heterologous proteins, EspA (E. coli secreted protein), UreB (H. Pylori urase subunit B) and hIL-24 (human Interleukin-24) -with different sizes and from different origin were selected to evaluate the extracellular secretion capability of the newly-developed pQABPS vector system. Results of Western blotting analysis revealed that they were efficiently secreted into culture supernatant.2) Extracellular expression product of recombinant hIL-24 was identified by ELISA. It showed that the secretion amount of hIL-24 was 336.5μg/L. The biological activity of inducing tumor cells apoptosis of recombinant hIL-24 was detected by MTT and morphology overview. It showed that the recombinant hIL-24 in the culture supernatant of M15 induced apoptosis of tumor cells in vitro, while and no effect on normal cells. It revealed that the hybrid hIL-24 expressed extracelluarly remained biological activity.3) The new host E. coli XL1-Blue harbouring pQABPS can also export the four heterologous proteins GFP, EspA, UreB and hL-24 to the outer space.4) Four plasmids lacking of rsaFb gene pQG2ABPS(-), pQA2ABPS(-), pQU2ABPS(-) and pQI2ABPS(-) were constructed and transformed to E. coli M15. Western blotting analysis revealed that the four heterologous proteins GFP, EspA, UreB and hL-24 were all secreted to the culture supernatant. There were no obvious change of secretion capacity.In conclusion, novel extracellular expression system, pQABPS/E. coli (M15), carrying Caulobacter crescentus RsaA secretion elements was developed. Caulobacter crescentus RsaA secretion elements and three other exogenous regulatory components were directionally inserted into pQE30 vector, generating the interested plasmid designated pQABPS. Four heterologous genes (gfp, espA, ureB and il24) were subjected to expression assays using this newly-developed expression system, pQABPS/E. coli (M15). Western blot analysis and bioactivity tests revealed that they were efficiently secreted into culture supernatant and remained biological activity, implying that pQABPS fulfills the capability of extracellular delivering bioactive foreign proteins in E. coli (M15). Taken together, the expression system of pQABPS/E. coli (M15) can function as another useful tool for extracellular secretary expression of exogenous proteins.