| Recently there is an increasing interest to study and apply the cell-free protein synthesis system in biology and biotechnology. Cell-free protein synthesis system enables the direct in-vitro expression of protein from template DNA or mRNA. This has been achieved by combining a crude lysate from growing cells, which contains all the necessary enzymes and machinery for protein synthesis, with the exogenous supply of essential amino acids, nucleotides, salts and energy-generating factors. Cell-free protein synthesis offers several advantages over conventional cell-based protein expression methods. For example: without cell wall and plasma membrane, the cell-free protein synthesis system is of full ability in gene transcription and protein translation, therefore, is favorable to synthesize proteins with some antibiotic properties. Furthermore, cell-free protein synthesis system can produce proteins directly from PCR linear templates without the need of constructing expression plasmids, allowing it to be easily adapted for high throughput protein synthesis, and to create a selection of very powerful tools for proteomic applications.Human defensins are a family of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which plays important roles in the defense against microbial invasion. Human defensins are hopeful to become new kinds of endogenesis antibiotics without acquired microbial resistance and has proven the ability in inhibiting the replication of HIV virus.In this work, the expression performance in E. coli cell-free system was compared by using two different HBD-2 genes. One was cloned by RT-PCR from human skin, and the other was synthesized chemically with preferential codons of hosts. The results revealed that, the expression level of target protein from the synthesized gene was the same to the cloned one.The expression level of HBD-2 under different fusion tags (trxA and GFP) was compared. The results indicate that the expression of small peptide is stabilized with the Trx-tag and GFP-tag in the cell-free system. The expression level of HBD-2 fused with GFP is lower than that fused with trxA, whereas, the GFP moiety provides directly visuable and quantitative monitoring of the polypeptide synthesis. Furthermore, our results also showed that, almost all of the HBD-2 fusion protein was expressed in soluble form in the E. coli cell-free system, whereas cell-based methods yield insoluble aggregates for many antibiotic peptides synthesis.The expression level under two different cell-free reaction mode (Batch-mode and Continuous Exchange Cell-Free (CECF) mode) was evaluated. The results indicated that about five-fold improvement of productivity (ca.2.0 mg/ml soluble fusion protein) could be achieved by using a CECF system compared to that in batch system. With continuoussupply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane, proteins can be synthesized continuously with longer period.A purification procedure was established to obtain the mature recombinant protein trxA-HBD-2 in a purity of 95.2% with a recovery of 50%. The fusion protein was cleaved by cyanogens bromide (CNBr), and the released mature HBD-2 had demonstrated strong inhibition on the growth of E. coli D31 at low concentration.The same expression strategy was used for the expression of HBD-3 and HBD-4 in E. coli cell-free system, respectively. This suggested that the cell-free system might be a preferable method for the production of proteins or polypeptides with antibacterial activity.In addition, we obtained 16 kinds of HIV target genes by PCR amplification, and constructed specific plasmids for the cell-free protein expression. Similarly, the PCR templates for cell-free protein expression were also constructed by using a two-step PCR amplification process. By targeting at one HIV protein-proteinase (P10) as a demonstrative protein, we used two expression templates (plasmid and PCR product) for P10 expression in the E. coli cell-free system. The results showed that the target protein P10 could be detected either by using plasmid template or by PCR template. This cell-free method can be used to express many HIV genes simulatenously, which will facilitate the construction of high-throughput screening models for new anti-HIV drugs in the future.Finally, the cell extraction from E. coli was prepared by ourselves. The cell growth phase, the pressure for cell disruption and the storage condition of cell extraction were optimized. In the meanwhile, the optimal substrate concentrations and the energy regeneration system were evaluated. Under the optimized conditions, the GFP reporter gene was efficiently expressed in the E. coli cell-free system.
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