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Les antibiotiques sont produits sous des formes pharmaceutiques telles que des pilules antibiotiques en ligne.

elles permettent d'injecter la quantité de préparation strictement nécessaire.


The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 Co-expression of chaperonin GroEL/GroES enhances in vivo folding of yeast mitochondrial aconitase and alters the growth characteristics of Escherichia coli Parul Gupta, Nishtha Aggarwal, Pragya Batra, Saroj Mishra, Tapan K. Chaudhuri Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India Received 24 January 2006; received in revised form 19 May 2006; accepted 23 May 2006 Available online 2 June 2006 Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However,preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins inE. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence ofrecombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependentgrowth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. colichaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of co-expressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroEL & GroES together compensate thisreduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 ◦C when compared with the M15 E. colicells, however, there is an increase of 20% at 37 ◦C indicating the participation of endogenous chaperonin in the folding of a fractionof over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cellswas enhanced by 30% at 37 ◦C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase.
Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.
2006 Elsevier Ltd. All rights reserved.
Keywords: Over expression of recombinant aconitase; In vivo protein folding; Escherichia coli growth profile; Bacterial chaperonin GroEL andGroES AMPR, ampicillin resistance; LB, luria broth; The production of recombinant protein in Escherichia APS, ammonium persulfate; DTT, 1,4-dithiothreitol; HEPES, 4-2- coli is one of the major efforts in biotechnology today. A hydroxyethyl-1-piperazineethanesulfonic acid; IPTG, isopropyl ␤-d- major limitation in the over expression of recombinant thiogalactoside; SDS-PAGE, sodium dodocyl sulfate-polyacrylamide proteins is the inability of many recombinant polypep- gel electrophoresis tides to fold into their biologically active conformations Corresponding author. Tel.: +91 11 2659 1012; fax: +91 11 2658 2282.
within the milieu of the bacterial cell. The question of E-mail address: (T.K. Chaudhuri).
protein folding has been a subject of intensive research 1357-2725/$ – see front matter 2006 Elsevier Ltd. All rights reserved.
P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 since wed that a denatured pro- of interest is a common method, making their down- tein could fold unassisted under in vitro conditions. In stream processing much easier. The main purpose of vivo protein folding is a different phenomenon, com- recombinant protein expression is often to obtain an plicated by macromolecular crowding in cytosol. The accumulation of high degree of soluble product in the mechanism of in vivo protein folding remains one of the bacterial cell. This strategy is not always accepted by most intriguing problems to be elucidated in molecu- the metabolic system of the host and in some situations lar biology The in vivo folding a cellular stress response is encountered pathways are affected by a number of factors, such as physico-chemical conditions of the cellular environment In the last decade, extensive amount of work has and transient interactions with other co translated pro- been done on the co-over expression of the GroEL/ES teins, not present in the simplified in vitro assays. In the in E. coli along with other foreign proteins. The co-over complex medium of the cell, the physical conditions of expression of the bacterial chaperone system GroEL/ES temperature, pH, etc., are restricted, and the concentra- along with several proteins like ␨-crystallin ( tion of macromolecules is high creating a dynamically malate dehydrogenase changing environment for newly synthesized proteins medium-chain acyl-CoA dehydrogenase (Molecular chaperones are a (MCAD) (carbamoylase class of proteins thought to facilitate protein folding in and aconitase sig- this environment ( nificantly enhance the yield of soluble protein. The effect of GroEL/ES on protein folding in association these helper proteins in the last decade has extended the with other chaperones like trigger factor field of in vivo protein folding enormously. As unfolded polypeptide contains many more exposed hydrophobic has also been studied. It residues than the polypeptide in its native state, they has been found that the over expression of GroEL/ES are much more susceptible to aggregation. Whether the restores appropriate protein folding in the cells where polypeptide is a nascent chain on a ribosome or a mature trigger factor and DnaK have been deactivated. Exten- protein recently unfolded due to stress, suppression of sive work on the mechanistic aspects of protein folding aggregation is essential in order to maintain proteins in presence of chaperonin has also revealed valuable in a state competent for folding. Molecular chaperones information on protein folding pathway and aggregation are large family of proteins found in all types of organ- isms and have a very important role in protein folding and maintaining protein homeostasis ( studies on the effect of co-expressing chaperonin in the Chaperone families are highly conserved across cell along with other proteins and their implications on genomes, suggesting that their functions are essential for the cell growth have not been thoroughly investigated.
cellular life (Chaperones are thought Parameters like optimum temperature, inducer concen- to prevent newly synthesized proteins from misfold- tration, duration of induction, etc., play an important ing and aggregating, impeding undesired hydrophobic role in enhancing the level of production of the desired interactions, and allowing alternative folding pathways protein in its native form. Here we have studied differ- (They bind to the exposed hydrophobic ent aspects of cell growth parameters during the over regions of nonnative proteins, hindering aggregation production of recombinant aconitase in presence and (Therefore, through regu- absence of over producing exogenous molecular chaper- lated cycles of peptide binding and release, chaperones onin, GroEL and GroES. Our main aim is to understand facilitate the acquisition of the active conformation of the conditions for optimum production of recombinant the polypeptides. The most extensively studied chaper- aconitase in the presence and absence of co-expressed ones are the chaperonin – GroEL and GroES – from chaperonin GroEL and GroES in E. coli and subsequent E. coli (The atomic struc- growth profiles and their temperature dependence.
ture of GroEL and GroES (are known and also that of the GroEL–GroES 2. Materials and methods
complex formed in the presence of ADP Over expression of the molecular chaperones in E. coli 2.1. Chemicals and reagents Luria broth (LB) for E. coli growth and antibiotics for obtaining large quantities of correctly folded protein kanamycin, ampicillin and tetracyclin were obtained P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 from HiMedia (India). 4-2-hydroxyethyl-1-piperazi- Expression was checked by running various sam- ples on 15% SDS-PAGE ( (DTT), acrylamide, bis-acrylamide, standard molecular To study the effect of temperature on weight markers, ammonium persulfate (APS) and the specific growth rate, the experiment was carried out isopropyl ␤-d-thiogalactoside (IPTG) were obtained at two different temperatures (25 ◦C and 37 ◦C). Effect from Bangalore, Genei (India). Other reagents and of induction on the growth rate of various strains was chemicals used were from Merck (Germany) and Sigma observed from the growth curve generated with and with- out induction.
2.2. Strains and plasmids 2.4. Determination of the specific growth rateconstants for bacterial growth The gene for yeast mitochondrial aconitase, cloned in the pQE60 vector from Qiagen (AMPR selectable The specific growth rate constants for various growth marker) with ColE1 origin of replication was obtained profiles was calculated by plotting absorbance versus from Dr. Sabine Rospert, Germany. The constructs time and obtaining the slope by exponential trend using pACYCEL over expressing GroEL and pACYCELS the following equation: over expressing GroEL and GroES (with tetracycline X = X resistance) are generous gift from Dr. Arthur L. Horwich, USA. M15 E. coli strain, K12 derivative was used for the where X = biomass at time ‘t'; X0 = biomass at time expression of various plasmids. M15 strain, containing ‘t = 0'; µ = specific growth rate constant; t = time in multiple copies of pREP4 plasmid, was maintained in presence of kanamycin. pREP4 plasmid carries the lacIgene encoding the lac repressor and hence the expres- 2.5. Estimation of the relative intensities of the sion of aconitase in pQE60 is regulated. M15 cells were bands in the SDS gel transformed with plasmid pAco to express only aconi-tase, with pAco and pACYCEL to express aconitase and BioRad (USA) gel documentation unit was used for GroEL, with pACYCELS to express GroE-GroES and estimating the relative quantities of the various protein with pAco and pACYCELS to express aconitase, GroEL bands observed on the gel. Gel image was taken and the and GroES. The antibiotic concentration used for the various lanes were framed using ‘manual frame lanes' optimum growth of the cells was 25 ␮g/ml, 80 ␮g/ml and toolbar. The number of lanes in the frame was kept the 12.5 ␮g/ml for kanamycin, ampicillin and tetracycline, same as that present in the gel. The lanes drawn were adjusted to fit the size of the lanes in the gel. Lane E. coli M15 strain was activated from a stab cul- background subtraction was carried out to remove the ture by streaking on luria agar plate supplemented with background intensity of the gel itself from the bands.
required amount of antibiotics. Ten milliliters LB sup- ‘Band analysis quick guide' from the Quantity one pro- plemented with antibiotics was inoculated and cultured gram (BioRad) was used to select the bands. Bands were at 37 ◦C. This strain was further maintained by mak- detected using ‘band detect' option and the area of the ing 20% glycerol stocks, frozen in liquid nitrogen and band with the required peak was adjusted using ‘adjust stored at −80 ◦C. These stocks were used subsequently band' option to get the region of the band to be estimated.
for making competent cells and transformation with var- Relative quantity of the band selected was measured by ious plasmids. Various transformed recombinant E. coli selecting ‘relative quantity' from ‘band attributes' tool- cells were also grown and maintained as described above.
bar. Relative quantity of a particular band is the quantitymeasured by its intensity, expressed as a percentage of 2.3. Determination of growth profile the total intensity of all the bands in the lane.
Various E. coli strains were grown at 25 ◦C and 2.6. IPTG titration 37 ◦C, at 200 rpm in shake flasks and 1 ml of cell sus-pension was withdrawn at various time intervals for Various E. coli strains transformed with recombinant turbidity measurements at 650 nm using UVIKON 930 plasmids were grown in LB medium supplemented with spectrophotometer (Kontron instruments, USA). Induc- antibiotics upto OD650 between 0.6 and 1.0. At this point tion was done at OD650 of 0.7–1.0 with 100 ␮M IPTG each culture was divided into nine parts of 10 ml each.
for expression of aconitase ( IPTG was added at nine different concentrations ranging P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 from 0 ␮M to 200 ␮M The assay was performed by taking 20–50 ␮g of The cultures were left overnight at 37 ◦C and the level protein in a 1 ml reaction volume (0.1 M Tris–HCl pH of expression were analyzed by 15% SDS-PAGE.
8, 0.66 mM sodium citrate, 0.66 mM MnSO4, 0.5 mg/ml ␤-NADP and 0.17 mg/ml isocitrate dehydrogenase). The 2.7. In vivo folding of aconitase formation of NADPH was monitored at 340 nm usingtime/kinetics application on Beckman Coulter DU 800 Amount of folded protein in a cell can be estimated based on the principle that the proteins with a three-dimensional structure are soluble in the cytoplasm and 3. Results
in aqueous buffer, however, denatured proteins are insol-uble and occur as aggregates 3.1. Changes in growth profile of E. coli cells on Thus, to estimate the extent of correct folding of aconi- transformation with various recombinant plasmids tase in vivo, the induced cells were pelleted and brokendown by sonication to release the intracellular compo- E. coli is used extensively for the expression and over nents in the lysis buffer. Normalization of the cell culture production of both prokaryotic and eukaryotic recom- was done, such that same number of cells were taken for binant proteins, as it requires very simple growth con- the analysis of each sample. The soluble components ditions. The growth profile of E. coli changes consider- were separated from the insoluble mass by centrifuga- ably depending on the type and number of recombinant tion of the cell lysate. The supernatant and the pellet plasmid it contains and the number of proteins it over were analyzed separately for the presence of aconitase by expresses. Bacterial cell growth was monitored by mea- SDS-PAGE and enzymatic activity test. Culture broths of suring turbidity of the cell culture at 650 nm using a different transformed strains expressing aconitase at dif- ferent temperatures were harvested and resuspended inlysis buffer containing 50 mM HEPES (pH 7.4), 0.5 mM 3.1.1. Effect of plasmid characteristics on the MgCl2, 1 mM DTT (These cells growth rate of transformed cells were disrupted using ultrasonicator, followed by cen- The growth curve for various transformed strains of E. trifugation at 10,000 rpm for 45 min. The supernatant and coli namely, M15 cells expressing aconitase, M15 cells pellet were separated and freeze dried in a lyophillizer expressing aconitase and GroEL, M15 cells expressing (LABCONCO freezedry 4.5). Lyophilized supernatant aconitase, GroEL and GroES and M15 cells expressing and pellet were resuspended in the loading buffer and GroEL and GroES were generated by plotting turbid- analyzed by SDS-PAGE. Aconitase activity assay was ity values at 650 nm of various strains against growth done taking 50 ␮g of total protein in each case.
time without induction. M15 strain was used as a neg-ative control in all these studies. The growth profiles of 2.8. Aconitase assay transformed cells without induction showed the effectof plasmid replication and maintenance on the rate of Aconitase activity was quantitated using a coupled growth At both the temperatures (25 ◦C and enzyme assay. Aconitase catalyses conversion of cit- 37 ◦C), cells containing pACYCEL plasmid showed a rate to isocitrate, which in turn is converted to ␣-keto decrease of more than 25% in the growth rate. At 25 ◦C, glutarate in presence of isocitrate dehydrogenase along presence of pACYCELS plasmid in M15 cells reduced with the formation of NADPH from NADP the growth rate by about 40% as compared to M15 wild Table 1Specific growth rate constants (µ) for the growth of M15 E. coli strain under various plasmid-containing situations at 25 ◦C and 37 ◦C E. coli strain µ at 25 ◦C µ at 37 ◦C Uninduced strains Uninduced strains M15 + Aco + GroEL M15 + GroEL + GroES M15 + Aco + GroEL + GroES Regression coefficient values are given in brackets.

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 type cells, whereas, at 37 ◦C, presence of pACYCELS presence of pREP4 plasmid in the M15 E. coli strain pro- plasmid showed an increase in growth rate by about 10% vides the lacI gene from the Lac operon, which represses No appreciable change in the growth pro- the aconitase expression in pQE60. Addition of inducer file was observed for M15 cells on transformation with to the cell culture medium activates the expression of the aconitase plasmid ( aconitase. A kinetics analysis, after addition of IPTG,was done to estimate the duration of induction required 3.1.2. Effect of IPTG induction on the growth for the expression of aconitase in presence and absence of profile of various transformed strains at different chaperonin at 25 ◦C and 37 ◦C. The expression of aconi- tase was estimated by SDS-PAGE analysis of samples Growth profiles of all the M15 strains transformed withdrawn at different time intervals (Figs. It with pAco, pAco-pGroEL, pGroELS, and pAco- was found that at 37 ◦C, aconitase expression in absence pGroELS were studied with and without IPTG induction.
of chaperonin requires 5 h of incubation after induction Induction was carried out with 100 ␮M IPTG in the tur- In presence of only GroEL it requires the least bidity range (OD650) of 0.6 to 1.0. At 25 ◦C, all aconitase time of 2 h for induction, whereas, when both GroEL and over expressing M15 strains, with and without recombi-nant GroEL/ES showed a reduction in the growth rate,the most notable however, are the M15 cells harboringonly aconitase, which showed a 30% reduction in growthrate At 37 ◦C, a complete reversal in the growthprofile was observed. Aconitase over expressing M15strains, showed an overall rise in growth rate after induc-tion. Only aconitase over expressing strain showed a riseof 20% when compared with wild type M15 cells and20% increase in growth rate was observed for aconitaseand GroEL over expressing strain (A signifi-cant rise of 30% was observed in the specific growth rateof M15 cells expressing aconitase in presence of bothGroEL and GroES. The changes in specific growth rateof various cultures on induction are unavoidable, how-ever, such changes can be minimized by redesigning andengineering various pathways 3.1.3. Effect of temperature on the growth profile ofvarious transformed strains after induction withIPTG Growth profiles of different transformed M15 cells containing pAco, pAco + pGroEL, pAco + pGroELS andpGroELS plasmids were studied at two different tem-peratures, 37 ◦C and 25 ◦C, after induction with IPTG.
At 37 ◦C, the growth rate for all transformed cells was Fig. 1. (A) Relative expression of aconitase with time in the presenceand absence of GroEL/ES at 37 ◦C. The graph shows the different found to be about two times higher than those at 25 ◦C for relative intensities of the aconitase bands in the gel depicting the lev- the same strain. Other changes in the trends for various els of expression of aconitase in presence and absence of GroEL and growth parameters for different transformed cells have GroEL/ES at 37 ◦C with different durations of incubations. The first been observed by changing temperature (as discussed in bar of the triad shows only aconitase expression, the second bar of the above result Sections the triad shows aconitase expression in presence of only GroEL andthe third bar of the triad shows aconitase expression in presence ofboth GroEL and GroES. (B) 15% SDS-PAGE shows level of aconitase 3.1.4. Over expression of aconitase in M15 cells expression in presence and absence of GroEL and GroEL/ES at 37 ◦C with time of induction in the absence and presence with different durations of incubations. Lane 2, 0 h; lane 3, 2 h; lane of over expressing chaperonin GroEL/ES 4, 5 h; lane 5, 7 h; lane 6, 9 h; lane 7, 11 h and lane 8, 15 h. Lane 1 The gene for yeast mitochondrial aconitase (pAco) is shows medium range standard molecular markers. Top panel showsover expression of aconitase only, center pane shows aconitase over cloned in pQE60 vector, which confers ampicillin resis- expression in presence of GroEL and bottom panel shows aconitase tance to the cells and has ColE1 origin of replication. The over expression of aconitase in presence of GroEL and GroES.

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 Fig. 2. (A) Relative expression of aconitase with time in the presence Fig. 3. (A) Optimization of IPTG concentration for aconitase expres- and absence of GroEL/ES at 25 ◦C. The graph shows the different sion. Variation of expressed aconitase with change in inducer con- relative intensities of the aconitase bands in the gel depicting the lev- centration. Solid squares show M15 cells expressing aconitase only, els of expression of aconitase in presence and absence of GroEL and solid triangles show M15 cells expressing aconitase and GroEL, solid GroEL/ES at 25 ◦C with different durations of incubations. The first circles show M15 cells expressing aconitase, GroEL/GroES. Opti- bar of the triad shows only aconitase expression, the second bar of mum IPTG required for induction of aconitase is 75 ␮M (Origin 5.0 the triad shows aconitase expression in presence of only GroEL and software was used to fit the graph). (B) 15% SDS-PAGE showing the third bar of the triad shows aconitase expression in presence of variation of expressed aconitase with change in IPTG concentration.
both GroEL and GroES. (B) 15% SDS-PAGE shows level of aconitase Lane 1, standard protein molecular weight markers. Lane 2, 0 ␮M expression in presence and absence of GroEL and GroEL/ES at 25 ◦C IPTG. Lane 3, 10 ␮M IPTG. Lane 4, 20 ␮M IPTG. Lane 5, 50 ␮M with different durations of incubations. Lane 2, 0 h; lane 3, 3 h; lane IPTG. Lane 6, 75 ␮M IPTG. Lane 7, 100 ␮M IPTG. Lane 8, 125 ␮M 4, 5 h; lane 5, 10 h; lane 6, 13 h; lane 7, 14 h and lane 8, 17 h. Lane IPTG. Lane 9, 150 ␮M IPTG. Lane 10, 200 ␮M IPTG. Top panel 1 shows medium range standard molecular markers. Top panel shows shows induced aconitase in absence of chaperonin, second panel shows over expression of aconitase only, center pane shows aconitase over induced aconitase expression in presence of GroEL, third panel shows expression in presence of GroEL and bottom panel shows aconitase induced aconitase expression in presence of GroEL and GroES and over expression of aconitase in presence of GroEL and GroES.
bottom panel shows constitutive expression of GroEL and GroES incells expressing no aconitase.
GroES are present, expression of recombinant aconitase for 24 h. No over expressed protein was found in M15 requires much longer induction period of 11 h. At 25 ◦C, cells on IPTG induction. GroEL and GroES were found expression of aconitase requires 13 h of incubation after to be over expressed constitutively and did not require induction in absence of chaperonin (). M15 cells any inducer for complete expression Recom- expressing aconitase and GroEL requires 10 h and aconi- binant aconitase expression was found to be inducible by tase expression in presence of both chaperonin requires IPTG. M15 cells expressing pAco gene in the absence 14 h of incubation after induction.
and presence of chaperonin was found to require 75 ␮MIPTG for optimum expression of aconitase 3.1.5. Inducer concentration required for optimumexpression of recombinant aconitase 3.2. Over expression of aconitase in E. coli M15 M15 cells transformed with pAco in presence and cells in presence and absence pGroEL/pGroELS absence of pGroEL/pGroELS were induced with dif-ferent concentration of IPTG, when the turbidity of the E. coli M15 strain containing pREP4 plasmid was culture at 650 nm reached to 0.9 and were left at 37 ◦C transformed with high copy number plasmid (pAco) con-

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 Table 2Expression level of aconitase in presence and absence of GroEL/ES at25 ◦C and 37 ◦C Relative quantity of Relative quantity of aconitase at 25 ◦C aconitase at 37 ◦C Aconitase with GroEL Aconitase with GroEL 3.2.1. In vivo folding of aconitase in absence andpresence of co-expressed GroEL and GroES in E.
coli cells
Non-native aconitase lodges itself as insoluble aggre- gates in a cell, which is deficient in chaperonins, ascompared to being fully soluble in a wild type cell pro-ducing both the chaperonin (Theextent of correctly folded native protein in a cell canbe determined based on the principle that, a folded pro- Fig. 4. (A) Expression levels of aconitase in the presence of GroEL and teins would be soluble, whereas the denatured proteins GroEL/ES. Graph shows changes in the level of expression of aconi-tase in recombinant M15 cells expressing aconitase in absence and would form aggregates and stay insoluble. On cell dis- presence of GroEL and GroES (relative band intensities of aconitase ruption and fractionation, the supernatant contains the have been compared in various cases). Bold check shows the varia- soluble proteins and all the aggregated proteins along tion at 25 ◦C and hollow check shows at 37 ◦C. (B) 15% SDS-PAGE with cell debris form a pellet. SDS-PAGE analysis of showing changes in the level of expression of aconitase in recombinant the supernatant and pellet fraction of the cell lysate M15 cells, expressing aconitase in absence and presence of GroEL andGroES. Standard molecular markers in lane 1 and lane 2 show unin- showed that, in the absence of exogenous duced aconitase, lane 3 and 4 show induced aconitase in duplicate, GroEL and GroES, ∼35% of the over expressed aconi- lane 5 show uninduced aconitase in presence of GroEL, lane 6 and tase was found in the soluble form in the supernatant at lane 7 show induced aconitase in presence of GroEL and lane 8 shows 25 ◦C which increased to ∼40% at 37 ◦C. In uninduced aconitase in presence of GroEL and GroES, lane 9 and lane presence of chaperonin GroEL, only 25% of the over 10 show induced aconitase in presence of GroEL and GroES.
expressed aconitase showed up as folded fraction inSDS-PAGE at 25 ◦C, whereas 20% of the over expressed taining the gene for the yeast mitochondrial aconitase aconitase appeared to be folded at 37 ◦C. When both with a Lac-regulated promoter. A distinct band in SDS- the chaperonin GroEL and GroES were present approxi- PAGE observed in the transformed strain clearly showed mately 50% of expressed aconitase was soluble at 25 ◦C the over expression of aconitase in the strain and about 40% at 37 ◦C Aconitase activity E. coli M15 strain containing pACYCEL plasmid con- data show that the soluble form of aconitase is stitutively over expressing GroEL was transformed with also biologically active. When pGroEL alone is present pAco and the expression of aconitase showed a marked in a cell, which is over expressing aconitase, aconitase reduction of about 40% at 37 ◦C Whereas, gets trapped in the hydrophobic cavity of GroEL; hence when pAco is expressed in presence of pGroELS the it neither folds nor gets released as a folding competent reduction in aconitase expression is only 20%. The intermediate. GroEL remains in the fully native form in presence of the gene for a large 800-kDa protein in the cell and hence appears in the supernatant. The pro- pGroEL/pGroELS, directs most of the energy produced teins that remain bound with GroEL will appear in the by metabolism in the cell for over production of GroEL, supernatant, even if they are not biologically active or resulting in a reduced aconitase expression in the cell.
in a folded state. Thus, some GroEL bound non-native Presence of both GroEL and GroES in the cell helps to aconitase appears in the soluble form with GroEL alone fold the over expressed aconitase correctly. At 25 ◦C, when tested by SDS PAGE (The increase in the almost no change in the expression in the aconitase is amount of folded aconitase in presence of GroEL and observed in presence of GroEL/GroES and GroES clearly shows that presence of both GroEL and GroES are required for the correct folding of aconitase.

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 These observations are consistent with the earlier obser-vations by aconitase does notfold correctly on inactivation of the chaperonins in yeastmitochondria.
Cells derive energy for growth from metabolic activ- ities. They use this energy to carry out essential func-tions like reproduction, growth, synthesis of various bio-molecules like proteins and nucleic acid, wear and tearmanagement, etc. When a cell is programmed to producelarge amount of one or more recombinant proteins, theinherent energetics of the cell growth gets disturbed. Theenhanced metabolic load exerted on the cell for main-tenance and expression of recombinant plasmid mayadversely affect the rate of growth of a cell produc-ing recombinant proteins as compared to the wild typestrain (Reduction in specific growth rates inpresence of a large number of plasmid in a recombinantcell is well known However, in our studywe found varying trends. Transformation of M15 E.
strain with pACYCEL/ES increases the growth ratesignificantly at 37 ◦C, whereas it decreases at 25 ◦C.
Higher temperature acts as an inducer for production ofheat shock proteins like GroEL and GroES. EnhancedGroEL/ES production at 37 ◦C helps the cells in pre-venting aggregation, which enhances the cell efficiencyand induces the correct folding of various proteins in thecells resulting in enhanced growth. At 25 ◦C, aggrega-tion of both native and recombinant proteins is much lessin comparison to 37 ◦C. Thus, at 25 ◦C, the advantageof over expressed GroEL and GroES becomes a liabilitywhere it has to spend a lot of energy for synthesis of large800 kDa GroEL (resulting inreduction of the growth rate in presence of chaperonin.
Only pAco containing cells seemed to have had a verylittle effect on the growth rates of uninduced cultures.
As M15 cells contain endogenous aconitase as well Fig. 5. (A) In vivo folding of aconitase in the absence and presence of as GroEL and GroES, hence, on transformation with GroEL/ES. Graph shows the change in the level of folded aconitase in pAco and pGroELS, the cells will produce recombinant the presence and absence of GroEL and GroEL/ES at 25 ◦C and 37 ◦Cin vivo. Bold waves show folding at 25 ◦C and hollow waves shows aconitase and chaperonin along with their endogenous folding at 37 ◦C. (B) 15% SDS-PAGE shows the change in the level counterpart. Enhanced production of heat shock proteins of folded aconitase in vivo, in the presence and absence of GroEL and at elevated temperatures in various organisms like E. coli GroEL/ES at 25 ◦C and 37 ◦C. First column of gels depicts folding at 25 ◦C and the second column shows folding at 37 ◦C. Total amount ofaconitase (whole cell) in the first lane, folded aconitase (supernatant) isseen in the central lane and aggregated aconitase (pellet) is in the thirdlane. Amount of folded aconitase in presence of GroEL and GroES can p, pellet). (C) Aconitase activity assay for over expressed aconitase the seen the most distinctly in the central panel. Aconitase in absence alone, with GroEL/ES and with GroEL alone are done and shown as of any chaperonin is shown in the top panel and aconitase in presence units per mg of protein per min. Black bars show activity at 25 ◦C and of only GroEL is in lowest panel (wc, whole cell; s, supernatant; white bars at 37 ◦C.
P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 parasites and brown This reduces the rate of protein synthe- are well documented. Endogenous chaperonin, sis (another ATP driven reaction) significantly. At lower being heat shock proteins are activated at higher tem- temperature the protein synthesis itself is very slow, peratures. Thus, on IPTG induction, all aconitase over requiring larger time for complete induction to occur.
expressing strain shows an increase in the growth rate For complete induction of the Lac–operon system a at 37 ◦C. The endogenous chaperones present in E. coli critical concentration of inducer is required to inactivate produced at higher rates at 37 ◦C may help a fraction of the repressor protein produced by lacI gene. Addition of the over expressed aconitase to fold correctly, which in IPTG binds to the active LacI repressor and causes dis- turn enhances the TCA cycle and generates more energy sociation from its operator. Use of IPTG as the inducer resulting in enhanced growth rate. Infact, the enzymatic for production of heterologous proteins has already been activity test reveals higher aconitase activity in pAco tested The optimum con- containing cells at 37 ◦C Co-expression of centration of inducer for aconitase expression was found GroEL along with aconitase at 37 ◦C showed a similar to be 75 ␮M. At lower concentration of IPTG the amount trend as that of only aconitase over expressing cells. A of aconitase produced was much less. Thus, varying con- major chunk of over expressed aconitase gets trapped centrations of the inducer can be efficiently used as a in GroEL cavity and in the simple tool for controlling the expression of recombi- absence of adequate GroES, it is unavailable for both nant aconitase in E. coli.
folding by active endogenous chaperonin and aggrega- Aconitase expression in E. coli seems to be affected tion. Growth rate is maximum when the entire folding by the over expression of GroEL and GroES. In pres- machinery is present since: (a) larger amounts of both ence of GroEL alone, the amount of over expressed GroEL and GroES help in correct folding of various aconitase reduces drastically. The reason may be that proteins, enhancing cell efficiency by minimizing toxic the cells are giving a priority to the synthesis and fold- effects of protein aggregation, and (b) the extra energy ing of the helper protein GroEL so that less energy is required for the expression and synthesis of GroEL and available for the synthesis of aconitase. In presence of GroES and for folding is off set by enhanced genera- both the chaperonin, the reduction in aconitase expres- tion of ATP by active TCA cycle due do increased active sion is almost negligible as the presence of the complete aconitase. Above results are also substantiated by cal- folding machinery increases the extent of correct folding culation of GroEL: aconitase ratio using relative band of newly synthesized aconitase. The excess ATP gener- intensities by gel documentation. An increase of 25% ated from enhanced TCA cycle through the participation in the GroEL: aconitase ratio was observed at 37 ◦C as of increased amount of correctly folded aconitase may compared to that at 25 ◦C in the cells over expressing serve two purposes: (a) enhancing cell growth rate and aconitase and GroEL.
(b) increasing rate of protein synthesis. The increase in Time of incubation required after induction to over protein synthesis can be seen in the form of enhanced expressed aconitase, for the pAco containing strain, in aconitase expression in presence of both GroEL and presence of chaperonin GroEL only, was found to be the least. Whereas, in presence of the complete fold- Reduced expression of aconitase in the E. coli cells ing machinery viz. GroEL and GroES the time required over producing GroEL also supports the observation that was the maximum as compared to the over expression of the least time of induction is required by cells over- aconitase in absence of chaperonin. Earlier reports ( expressing aconitase and GroEL simultaneously. Due to high expression of aconitase in strains expressing the that aggregate formation in cell causes reduction in cell entire chaperonin machinery—GroEL and GroES, the growth, inhibition of transcription and loss in cellular time of induction was very high. A large portion of the functions. Over production of aconitase only may lead cellular energy is involved in the folding process, result- to formation of insoluble aggregates in the cell, resulting ing in a slower rate of protein synthesis and hence, a in reduced cell efficiency. GroEL has the property to trap higher time of induction.
non-native aconitase, and preventing its aggregation in Enhanced efficiency of correct folding of aconitase cell enhancing efficiency of cell function, requiring less in E. coli cells and the maximum percentage of native time, of incubation after induction. In presence of exoge- aconitase was found in the case when both GroEL and nous GroEL and GroES, a large portion of energy in the GroES chaperonin were co-expressed along with aconi- form of ATP is used up for correct folding, as chap- tase at 25 ◦C. Even though, the rate of protein synthesis is eronin assisted protein folding is ATP driven reaction much higher at 37 ◦C as compared to 25 ◦C, the extent of P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1975–1985 folding is higher at 25 ◦C. The higher rate of aggregation Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., reaction at 37 ◦C competes with the chaperone assisted Horwich, A. L., et al. (1994). The crystal structure of the bacterial folding reaction, resulting in the loss of a large portion chaperonin GroEL at 2.8 A. Nature, 371, 578–586.
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Remove the muzzle and give rule 37(b) teeth: advocating for the imposition of sanctions for rule 26(c) protective order violations in the eleventh circuit

Remove the Muzzle and Give Rule 37(b) Teeth: Advocating for the Imposition of Sanctions for Rule 26(c) Protective Order Violations in the Eleventh CircuitAmber M. Bishop Follow this and additional works at: Recommended CitationAmber M. Bishop (2014) "Remove the Muzzle and Give Rule 37(b) Teeth: Advocating for the Imposition of Sanctions for Rule 26(c)Protective Order Violations in the Eleventh Circuit," Georgia State University Law Review: Vol. 31: Iss. 2, Article 5.Available at:

The welfare effects of innovative pharmaceuticals

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