In vitro ekspresjonssystem for produksjon av bakteriosiner og immunitets proteiner

Abstract

The structures of the immunity proteins for the two-peptide bacteriocins have not been determined, although knowledge of these structures is of great interest as it may reveal at a molecular level how these proteins render bacteria resistant to bacteriocins. Production of these immunity proteins in amounts required for structure studies has been hampered by their apparent toxicity to the producer-cells. A cell-free system for protein expression is an in vitro system for protein synthesis that provides a method for production of high amounts of protein required for structure characterization. The system allows for milligram amounts of protein or peptide to be produced without considering growth requirements of, or toxicity to the recombinant host-producer cell. It can be optimized for individual proteins, and is an inexpensive and efficient method. In this study, vector constructs, each containing a gene for one of three immunity proteins or one bacteriocin has been produced, to enable transcription of these genes followed by translation of their mRNAs into proteins. The cell-free expression system that was established in this master thesis is based on the method described by Pedersen et al. [1]. The bacteriocin lactococcin A (LcnA), the plantaricin EF immunity protein (PlnEFim), Enterocin 1071 immunity protein (Ent1071im), and the lactococcin G immunity protein (LcnGim) are all produced by lactic acid bacteria: Lactococcus lactis subsp. cremoris LMG 2130; Lactocbacillus plantarum, strain C11; Enterococcus faecalis strain FAIRE309; and Lactococcus lactis strain LMGT 2081 respectively. The genes encoding these proteins were introduced into an expression vector by combining techniques like PCR, TA-cloning and cloning by use of restriction enzymes. The expression vector pIVEX 2.4d contains a T7promoter for transcription and a His tag (histidine) to enable purification of the expressed protein from solution. T7RNA polymerase was isolated from a high yield producer, E. coli BL21 Star (DE3) with the Targe Tron® vector pAR1219 inserted, and used for transcription of the genes from DNA to mRNA. The translation machinery for protein expression was supplied by preparation of E. coli S12 cell extract, to be used in the cell-free system. In order to optimize the expression of these proteins, the effect of altering expression conditions, such as extract buffers and expression time were studied. The LcnA and LcnGim genes were successfully transcribed by the purified T7RNA polymerase and the LcnA protein may have been expressed in the cell free system, although the protein identity needs to be confirmed.