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Construction of efficient expression vector system using cis-acting elements for Lactococcus lactis

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농업생명과학대학 농생명공학부
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서울대학교 대학원
Lactic acid bacteriacis-acting elementpromoter modificationtuf
학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최윤재.
Many approaches have been attempted to improve the heterologous protein in various bacteria. Specifically, the plasmid-based expression system has been used to achieve the recombinant protein production as an easy and useful tool to manipulate. There are three strategies to improve the expression level of recombinant protein. Such as 1) introduction of high copies plasmid-based expression vector system, 2) construction of gene multimerization cassette as a insert and ligation with the backbone vector, and 3) search of new strong promoter. However there are several limitations for these strategies in that it is hard to replicate the DNA, is too large to transformed which causes genetic instability, and is hard to predict promoter strength. In this study, I modified the promoter region and tested on the promoter strength. In addition, I tried to introduce another cis-acting elements such as a transcriptional terminator and RBS (ribosome binding site) to improve the expression of recombinant protein.
Lactococcus lactis subsp. lactis IL1403 is widely used in the dairy and animal industries, and it is also studied for a live oral vaccine product to elicit mucosal immune response. The translational elongation factor Tu (tuf) gene is a house-keeping gene, and tuf promoter is characterized as a strong promoter in IL1403. In this study, tuf promoter was modified to test the efficiency of protein expression using the luciferase gene as a reporter.
Firstly two terminators, TrrnB and TpepN were tested for the luciferase gene expression efficiency. TpepN terminator showed better performance in luciferase expression.
Next, series of tuf promoter modification were attempted. In bacteria, RNA polymerases and several sigma factors recognized and recruited approximately -35 and -10 region upstream from the transcription start site. The core region including -35 and -10 hexamers in tuf promoter (119 bp) was amplified and series of modified tuf promoters were constructed using PCR with partial complementary reverse primer. There PCR products (#1) were cloned into the promoterless pIL.Ptuf.Luc(X) vector. Luciferase activity of t2, t4, t6 and t7 were higher than control tuf promoter. Especially t2 and t4 showed better performance, thus selected for next experiment.
It is well known that the sequence between RBS and start codon (ATG) are important for protein translation efficiency. Thus, I modified original sequence of this region, 'CATTTTTCAT' to 'AATTTTTAAA' to give more AT-rich. This modification was combined with selected modified tuf promoter to give a series of new tuf promoter cassette. The transformed IL1403s containing modified promoter (#2) were assayed for luciferase activity. Derivative of t2 and t4, t2-1 and t4-1 showed better performance.
Combined all the modified clones, luciferase activity was compared. t4-1 showed much higher activity compared to the t4, indicating the sequence between RBS and start codon is important for protein expression. To confirm this results, luciferase expression was analyzed on SDS-PAGE and western blot assay. Luciferase bands (61 kDa) was not detectable in SDS-PAGE, but in western blot, clones with t2, t4, t4-1 showed stronger signal compared to original tuf one.
In conclusion, this study revealed that introduction of modified strong promoter and additional cis-acting elements can improve the protein expression in IL1403. And this strategy has a prospect to improve recombinant protein expression. Since pIL252 is a low copy plasmid-based expression system, high copies-based plasmids are needed to increase recombinant protein expression.
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