Vibrio fischeri luxR Gene과 Promoter를 이용한 대장균 내에서의 새로운 단백질 발현 시스템

Robert J. Mitchell, 구만복*

광주과학기술원, 환경공학과, *mbgu@kjist.ac.kr

A Novel Protein Expression System for Escherichia coli

Using the Vibrio fischeri luxR Gene and Promoter

Robert J. Mitchell and Man Bock Gu*

Dept of Environmental Science and Engineering,

Kwangju Institute of Science and Technology, *mbgu@kjist.ac.kr

Several recombinant plasmids and strains were constructed.  Each of the plasmids has either the promoter from Vibrio fischeri's lux operon or the lac promoter controlling expression of the Xenorhabdus luminescens lux operon.  The strains, depending on their promoter, were induced with the autoinducer of V. fischeri or IPTG. The expression of these plasmids was monitored under various growth conditions and amount of autoinducer added and were compared in terms of their luminescence and protein levels in flask cultures.  Expression from the lac promoter showed a constitutive expression and reached a maximum of around 80,000 A.U. (Arbitrary Units) when not induced.  The strains bearing the V. fischeri promoter showed a low expression level, typically below 50 A.U., until induced, after which the bioluminescence level increased to give an induction of greater than 50,000-fold over the control.

Introduction

The use of recombinant proteins that are expressed in bacteria are commonplace in bio-industry.  One typical and well known method for such over-expression is the use of the lac promoter and the addition of isopropylthiogalactoside (IPTG).  The expression of the recombinant protein is increased tremendously in the presence of IPTG as the lac promoter is derepressed and RNA polymerase can bind to the strong promoter.  The promoter from V. fischeri is also an inducible promoter.  However, its induction is accomplished by a molecule made by the bacterium itself.  This molecule is called an autoinducer (A.I.) as it induces its own production.  Figure 1 shows the process responsible for the production of autoinducer and bioluminescence (BL) in V. fischeri.  As with the lac promoter, the Vibrio fischeri promoter has a basal level expression even in the absence of the autoinducer or where low concentrations of autoinducer are present.  When the A.I. reaches a certain concentration, it preferentially binds to the LuxR protein and this complex binds to the operator region of the promoter, by which it aids in the initiation of RNA polymerase.  The result is its own gene's production as well as the genes required for bioluminescence.

Several strains with this promoter controlling the expression of a luciferase from another bioluminescent bacterium were constructed to determine the induction kinetics and the possibility of using the promoter and the LuxR protein in the over-production of recombinant proteins.  In each of these strains, the luxI gene is truncated and non-functional and so they require the addition of autoinducer for high level production of bioluminescence.

Materials and Methods

Bacterial Strains and Plasmids

Plasmid pSB401 and pSB403 were received from Wilson et al and both contain the promoter region and luxR gene from V. fischeri controlling expression of the X. luminescens lux operon.  Plasmid pSB401 was transformed into E. coli strains RFM443 and JM109 to give BM14 and 401-JM, respectively.  Plasmid pDEW201 has a multiple cloning site in front of the X. luminescens operon on a pBR322 vector.  The V. fischeri promoter region was removed from pSB403 using an EcoRI digest and ligated into the MCS region of pDEW201 to give pDEWLuxR. After ligation, the plasmids were transformed into E. coli strain W3110.  Colonies were checked for bioluminescence in the presence of autoinducer.  Two colonies were selected, one was very bioluminescent and was labelled as BM29-CH and the other as BM30-B.  GC2 is an RFM443 strain harboring the pLITE201 plasmid (lac::luxCDABE).  The autoinducer used in this study was produced by BM2, RFM443 bearing plasmid pBLH105.  This strain was grown overnight in TB broth after which the culture was centrifuged and subsequently filter-sterilized.

Growth Conditions and Bioluminescence Measurement

All strains were grown at 37℃ in a shaking incubator, 250 rpm.  Overnight pre-cultures were grown in 100ml Luria-Bertani broth with appropriate antibiotics.  The test cultures were then inoculated with 0.1ml or 1ml of this overnight culture.  As the culture grew, autoinducer was added to a concentration of 0.05, 0.1, 0.5, 1, 2 or 5 % (v/v).  The O.D. was measured using a spectrophotometer at 600 nm.  Bioluminescence of the cultures was measured by removing 0.1ml to a luminometer.  In the cases were the luminescence exceeded the maximum light level of the luminometer, it was diluted using pre-warmed media.

Results and Discussion

Figures 2(a and (b show the induction of BM14 and 401-JM in the presence of autoinducer and Figure 3 shows the growth and expression of GC2 without IPTG addition.  It is very apparent that even without addition of inducer to any of the strains that the pLITE201 strain, GC2, has a greater basal level expression.  After prolonged growth, i.e. overnight, GC2 has a bioluminescence value of greater than 70,000 A.U. even without IPTG addition while both 401-JM and BM14 have an expression level of less than 200 A.U. (Data not shown)  For these two strains, however, Figure 2 shows that the addition of autoinducer media results in the induction of bioluminescence.  For BM14, there seems to be a repression of the induction until an O.D. of about 0,8 - 0.9.  For 401-JM, this is seen at an O.D. of around 0.25 - 0.3.  It is thought that this repression is due to action of intra-cellular mechanisms, which may include HNS production, lexA repression of the promoter, catabolite repression and possible luxR degradation by lon protease.  However, the exact reason for these differences is not known.  The kinetic curve for the rate of bioluminescence induction versus the volume of autoinducer added shows a typical Michaelis-Menton relationship (Data not shown).  Using the values from the experiments, it was found that the greatest rate of SBL change, i.e. Vmax, is about 4000 A.U./minute and the Michaelis constant, Km, in terms of the amount of autoinducer media added is about 2.54 mL.

The W3110 strains showed no repression.  Figure 4 shows the expression of BM30-CH.  It is obvious that the BL increases even with the very low optical density (< 0.01) and continues to increase in a logarithmic fashion.  As well, the final expression level was nearly 106A.U., approximately a 50,000-fold increase over the control.

These results show the potential application of this system in the production of a recombinant protein.  With an induction of nearly 50,000-fold in BM30-CH and even greater in BM14 (Data not shown) and the fact that the autoinducer molecule is a naturally produced substance, allowing for a high level production with minimal costs, this system offers a competitive alternative to the use of IPTG for the over-production of recombinant proteins.  As well, the concern with basal level expression of proteins

toxic to the bacterium is always a concern.  This study shows that the use of the V. fisheri promoter/luxR system offers a more viable option than the use of the lac promoter as the stringency of this system is tighter and can be controlled further through catabolite repression (Data not shown).

Acknowledgements

This research was supported by the KOSEF through ADEMRC at K-JIST. Authors appreciate their support.

References

Hanzelka, B., Greenberg, E.P. 1996. Quorum Sensing in Vibrio fischeri: Evidence that S- Adenosylmthionine Is the Amino Acid Substrate for Autoinducer Synthesis. J. Bacteriol. 178:5291-5294

Winson, M.K., Swift, S., Fish, L., Throup, J.P., Jorgensen, F., Chhabra, S.R., Bycroft, B.W., Williams, P., Stewart, G.S.A.B. 1998. Construction and Analysis of luxCDABE-Based Plasmid Sensors for Investigating N-Acyl Homoserine Lactone-Mediated Quorum Sensing. FEMS Microbiology Letters 163:185-192

LuxR	A.I.	BL

LuxI

Low [A.I.]    luxR promoter	luxI	luxC	luxD	luxA	luxB	luxE

High [A.I.]

Figure 1. Simplified mechanism of the Vibrio fischeri promoter

Figure 2. Induction of bioluminescence with different volumes of autoinducer media.  a) BM14  b) 401-JM  Volumes of autoinducer media added are listed in the legend and are in mls.

Figure 3. Growth and luminescence    Figure 4. Growth and luminescence  of GC2	of BM30-CH