Methods For Prokaryotic Expression Of Active Proteins And Peptides

With the rapid development of genomics and proteomics,the biological sequence database contents are exponentially increasing and accordingly molecular biotechnology including molecular cloning and heterologous expression has got unprecedented progress.In this article,several methodological improvements were made in respects of vector construction,heterologous protein or peptide expression and purification,which would promote high-throughput structure/function relationships study and further exploitation of the novel proteins or peptides.Firstly,we established a seamless gene cloning method mediated by mega primer and PCR,named MP cloning,by which not only long DNA fragments could be efficiently inserted into but also multiple fragments could be assembled into the plasmid vectors.Two sequential PCR steps were needed: target DNA fragments were amplified in the first round PCR,and the fragments were used as mega primers to amplify the total length of the plasmid vector.After DpnI digestion,the PCR products could be transformed into the E.coli competent cells.In addition to its efficiency and simplicity,the method is cost-effective,independent of expensive enzymes such as recombinase,T4 DNA polymerase or Type IIS restriction enzyme.MP cloning is proved to be an efficient tool for vector construction.Using seamless cloning methods,we successfully constructed pET-GST-SUMO-toxin expression vector.By the exression system,three peptide toxins including rHNTX-I,rHNTX-IV and rHWTX-Xa were produced in E.coli.It was demonstrated that rHNTX-IV has similar activity to native HNTX-IV by electrophysiological experiments.On the other hand,taking advantage of the hydrophobic differentiation between toxin peptides and proteins,we developed a peptide purification method based on TCA precipitation.The proteins could be efficiently precipated while most of the peptides existed in the supernatant.Peptides with high purity could be harvested by RP-HPLC.It was suggested that the method is more suitable for production of hydrophilic,stable peptides.Trx-6×His-SUMO tag combined with Shuffle strain as host is a more robust system for the disulfide bond formation.The purification conditions of immobilized metal affinity chromatography(IMAC)are mild.By this system,we successfully expressed and purified rTX-6868 and rHWTX-XVIIa.The system is proved to be more efficient for proteins,hydrophobic peptides or peptides with multiple disulfide bonds.Gene synthesis is a useful tool for acquisition of the coding DNA of target proteins or peptides.In this study,we established a single-tube reverse PCR(STRP)based gene synthesis method,which is very simple and efficient.The major advantage of STRP method is that gene synthesis and vector construction can be accomplished in one round of PCR,regardless of cumbersome sub-cloning steps.By STRP,three genes around 700 bp have been synthesized and successfully expressed in E.coli.Till now,the longest gene that can be synthesized by STRP is up to 1400 bp.Errors introduced by oligonucleotides can be easily corrected by overlap PCR.Application of STRP gene synthesis method in the prokaryotic expression platform can dramatically promote DNA manipulation and shortened the period of protein expression.In conclusion,molecular cloning and heterologous protein/peptide expression and purification as fundamental techniques in almost every laboratory,improvements in them can promote the whole project.In this artical,we made several improvements and innovations in these methods.These methods could be complements to the routine laboratory techniques and provide important references for researchers.