Expression and translocation of glucose isomerase as a fusion protein in E. coli

Enzyme and Microbial Technology 35 (2004) 105–112

Expression and translocation of glucose isomerase as a fusion protein in E. coli Berna Sarıyar1 , Pınar Özkan2 , Betül Kırdar, Amable Hortaçsu∗ Department of Chemical Engineering, Bo˘gaziçi University, Bebek, 34342 Istanbul, Turkey Received 3 January 2003; accepted 4 October 2003

Abstract Glucose isomerase of Thermus thermophilus was fused to maltose binding protein to use its signal sequence and slow folding characteristic for transport to the periplasm in Escherichia coli. The product was mostly retained in the cytoplasm and 1.6% of the total glucose isomerase activity was detected in the periplasm as a fusion protein. The effect of inducer concentration on translocation was insignificant, however induction at 23 ◦ C increased periplasmic glucose isomerase fusion by 50%. Growth medium was supplemented with amino acids to investigate their effect on translocation. Addition of 0.5% (w/v) alanine, the most abundant amino acid in the glucose isomerase sequence, increased expression by 24%, and induced extracellular secretion of the fusion protein by 18%. On the other hand, glycine retarded growth and caused lysis. The elevated pH of cultures with alanine indicated its possible effect of on translocation, but no significant change was observed by externally imposed pH variations. These results indicate that the secretion efficiency of a fusion protein depends on the characteristics of the system used. © 2004 Elsevier Inc. All rights reserved. Keywords: Translocation; Fusion protein; Glucose isomerase; Maltose binding protein

1. Introduction Due to the wealth of genetic and physiological information available, Escherichia coli is the most versatile host for the production of recombinant proteins. However, most of these proteins are retained in the cytoplasm where they are synthesized. Although it is usually desirable and sufficient to achieve maximal production within the cytoplasm, it might be advantageous to target the recombinant protein to noncytoplasmic locations. In E. coli, periplasm is a favorable location as the final destination of recombinant proteins. Translocation to the periplasm requires the passage through a single membrane, the inner membrane only. Most importantly, foreign proteins are commonly soluble and biologically active in this compartment [1]. Purification of periplasmic proteins requires less complicated methods than cytoplasmic proteins. Traditional methods of protein recovery form the cytoplasm which require ∗ Corresponding author. Tel.: +90 212 3596469; fax: +90 212 287 2460. E-mail address: [email protected] (A. Hortaçsu). 1 Present address: Department of Chemical Engineering, Marmara University, Istanbul, Turkey. 2 Present address: Department of Molecular Biology and Genetics, Haliç University, Istanbul, Turkey.

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2003.10.021

cell disruption by either mechanical, physical, or chemical means [2] can cause protein degradation due to shear or heat, and chemical-mediated inactivation. There will also be difficulties in protein purification due to contamination [3]. On the other hand, periplasmic proteins can easily be released from the cell by osmotic shock [4]. Export of recombinant proteins to cell culture is even more favorable. Proteins can simply be purified from the culture broth after precipitating the cells by centrifugation. However, in E. coli, translocation to external milieu requires the passage of proteins through two concentric membranes, the inner and outer membranes. Therefore it is more complicated and difficult. In recent years, several hybrid systems have been constructed to develop simple purification methods. This approach uses large peptides or proteins as the fusion partner. As well as enabling translocation to non-cytoplasmic locations, this method enables purification of the fused protein by one-step affinity chromatography. The disadvantage of the fusion partner is that it must usually be removed for a well-characterised activity. The decision for the best fusion system for a specific protein of interest depends on the target protein itself (e.g. stability, hydrophobicity), the expression system, and the application of the purified protein [5].

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The objective of this work was to study the expression and translocation of glucose isomerase (GI) in E. coli. GI (EC 5.3.1.5) of Thermus thermophilus [6] is a thermostable cytoplasmic enzyme, which reversibly catalyzes the isomerization reaction between d-glucose and d-fructose. GI is an important commercial enzyme in industrial production of sweet high fructose syrups that are used as alternative sweeteners to sucrose or invert sugar in the food and beverage industry [7]. One of the improvements for large-scale production would be a lower-cost enzyme recovery from the host cells after fermentation [8]. For our study, maltose-binding-protein (MBP) fusion system was chosen to express GI as a periplasmic fusion protein. This would enable purification of GI directly from the periplasm. Amylose affinity column could further facilitate purification of the MBP-GI fusion protein. The results indicated that although GI was successfully expressed with the chosen system, there was a limited success in terms of translocation even with expression at low temperatures and amino acid supplementation. This might be due to steric factors and kinetic properties of the protein, as well as the cleavage of the fusion partners prior to translocation.

2. Materials and methods 2.1. Bacterial strains and plasmids Thermus thermophilus strain HB8 (ATCC 27634) was purchased from ATCC (USA). E. coli TB1 [F− , ara,
(lac-proAB), rpsL (Strr ), ␾80dlacZ
M15, hsdR (rk − , mk + )], E. coli ER2508 [Ion::
16
17,
(malB)zkb::5,
(lacZYA-argF)U169,
(mcrC-mrr)20, ara-14, galK2, rpsL20, xyl-5, mtl1, supE44, leuB6, fhuA2], and pMAL-p2 and pUC18 plasmids were supplied by the manufacturer New England Biolabs (Beverly, MA, USA). E. coli strain ER2508 strain has a partial deletion of the malE gene. E. coli strain DH5␣ was provided by Dr. Peter Ray from Hospital for Sick Children Toronto, Canada. Recombinant plasmid pPGI, containing the GI gene, was provided by Pinar Ozkan.

gene of E. coli, which encodes MBP. Expression was under the control of tac promoter and the malE translation initiation signals. GI gene was excised from pPGI by PstI digestion and cloned into pUC18 to make pPBGI. These expression plasmids were transformed into E. coli strains by electroporation [9]. All basic recombinant DNA techniques were carried out as described by Sambrook and co-workers [10]. 2.3. Cultivation media and conditions Cells were grown in shaking culture (180 rpm, 37 ◦ C) in LB broth containing 100 ␮g/ml ampicillin. Concentrated solutions of amino acids were autoclaved separately and added to LB broth at the time of inoculation. Protein expression from pBGI and pPBGI were induced by isopropyl-␤-d-thiogalactopyranoside (IPTG) addition (0–20 mM). These cells were incubated for 25 h. For expression at lower temperatures, incubation temperature was shifted down to 30 ◦ C or 23 ◦ C at the time of induction. 2.4. Preparation of cell extracts For the assay of extracellular GI, cells in the culture broth were pelleted and the proteins in the supernatant were precipitated by (NH4 )2 SO4 at 70% saturation (0 ◦ C). Proteins were pelleted by centrifugation at 26,000 × g for 30 min and resuspended in 50 mM MOPS to be assayed. GI was purified form the periplasm using osmotic shock, a variation on a published protocol [11]. Briefly, cells were pelleted by centrifugation at 9000 × g for 8 min and then resuspended in 2% of the original culture volume of 20% glucose in 30 mM Tris–HCl, 1 mM EDTA, pH 8. Cells were pelleted and resuspended in ice-cold 5 mM MgSO4 . The cells were then stored on ice for 30 min and pelleted. Supernatant containing the periplasmic proteins was assayed for GI activity. Pellet removed from the osmotic shock fluid was suspended in 10% original culture volume of 50 mM, pH 7.0, MOPS and subjected to sonication. Cells were pelleted and the supernatant was assayed for cytoplasmic GI activity. 2.5. Enzyme assay and protein analysis

2.2. Cloning of the GI gene GI gene (1183 bp) [6] was amplified from T. thermophilus genome by PCR using VentR DNA polymerase with the oligonucleotides BGIF (5 -GTG GAA TTC TGT GTA CGA GCC CAA ACC GGA-3 ) and PGIR (5 -GTG CTG CAG GCC GAT GGC CGC CCT CAC C-3 ) and digested with EcoRI and PstI. Prior to PstI digestion, EcoRI digested site was treated with the Klenow fragment of DNA polymerase to fill in the hangover sequence to make an XmnI site. This PCR product, verified by DNA sequencing, was cloned into pMAL-p2 to make the periplasmic expression vector pBGI. The amplified gene was inserted downstream from the malE

Equal volume of cell extract and reaction mixture (0.8 M glucose, 10 ␮M MgSO4 and 1 ␮M CoCl2 ) were mixed and allowed to react for 30 min at 65 ◦ C [8]. Enzyme reaction was stopped on ice. The amount of fructose formed was estimated by the cysteine–carbazole–sulfuric acid method [8,12]. One unit of activity was defined as the amount of enzyme which released 1 ␮m of ketose per minute under the assay conditions [8]. Protein concentrations were determined by the method of Bradford [13] using bovine serum albumin as the standard. SDS-PAGE of proteins was carried out according to the method of Laemmli [14].

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2.6. Purification of MBP-GI The MBP-GI fusion protein was purified by affinity chromatography using amylose affinity resin as described by the manufacturer, BioLabs.

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Table 1 Cytoplasmic enzyme activity in different fractions of the affinity column (1 mM IPTG) Fraction assayed

GI activity (U/L culture)

Cytoplasm (crude extract) Eluate from the column Flow through

1595.5 824.3 319.2

3. Results and discussion 3.1. GI production E. coli TB1 and ER2508 cells harboring the construct pBGI when induced with IPTG express MBP-GI fusion protein. MBP is a periplasmic protein translocated through the cytoplasmic membrane by the Sec machinery [15,16]. This experimental study was undertaken to explore the possibility of translocation of MBP-GI fusion to the periplasm by the presence of the signal peptide and the slow folding characteristic of the preMBP sequence. E. coli cells were induced with 0.5 mM IPTG and cytoplasmic, periplasmic and extracellular extracts were assayed for MBP-GI expression on SDS-PAGE. The expected fusion has a molecular weight of 86.7 kDa, of which 44 kDa is GI [6] and 42.7 kDa is MBP [17]. Analysis of the cytoplasmic extracts from uninduced (Fig. 1, Lane A) and induced (Fig. 1, Lane B) recombinant TB1 cells on SDS-PAGE indicated that there is a strongly induced band of 87 kDa in the induced cells. MBP-GI in the cytoplasmic extract of the induced sample was purified by amylose affinity column and analysed on the same SDS-PAGE (Fig. 1, Lane C). As well as the band for MBP-GI, a second band appeared in the eluate from the column. This band corresponded to MBP (42.6 kDa). GI activity in various eluate fractions and the crude extract are given in Table 1. The results showed evidence of the presence of GI in the flow-through material. This indicated that some MBP-GI was processed and GI flowed down the column in the flow-through material.

Fig. 1. SDS-PAGE analysis of total cell extracts of E. coli TB1(pBGI): (A) extract from uninduced sample; (B) extract from cells induced with 0.5 mM IPTG; (C) eluate after extract from (B) was applied to amylose column.

The analysis of the periplasmic extracts from TB1 and ER2508 strains on SDS-PAGE (Fig. 2) showed a faint band corresponding to MBP-GI. Enzyme assay analysis verified that this band was indeed active MBP-GI. The lower relative yield of the periplasmic fusion protein obtained in this study is comparable to the results from the expression of other proteins using the pMAL-p2 vector [18]. In order to understand the source of the strong band for MBP in the periplasmic extract of TB1 cells (Fig. 2, Lane A), MBP-GI expression was compared inTB1 and the MBP deficient strain ER2508. SDS-PAGE of the extracts from this strain showed the absence of this much MBP in the periplasm (Fig. 2, Lane B). This indicated that most of MBP in the periplasm of TB1 cells was produced by the host cell. It was not from processed MBP-GI in the periplasm. The latter could only be found in negligible amounts. 3.2. Effect of IPTG concentration on GI production The effect of IPTG concentration on recombinant protein production and translocation in TB1 cells was investigated, and the results are shown in Table 2. There was no detectable activity in the uninduced culture, but as the IPTG concentration increased, the total GI activity gradually increased. However the relation was not linear, extrapolation of the data in Table 2 suggested that total GI activity started to approach a saturation value of 2300 units per liter for inductions above 3 mM IPTG. The specific activity of GI also increased with increasing IPTG concentrations. Comparison of the cytoplasmic GI of the culture induced with 0.5 mM IPTG with the cytoplasmic GI of the culture induced with 1.0 mM IPTG showed that the concentration of the recombinant protein was 50% more for the culture induced with 1.0 mM IPTG. However when the periplasmic

Fig. 2. SDS-PAGE analysis of periplasmic extracts of cultures induced with 0.5 mM IPTG: (A) osmotic shock fluid from E. coli TB1(pBGI) cells and (B) osmotic shock fluid from E. coli ER2508(pBGI) cells.

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Table 2 The effect of different inducer concentrations on enzyme activity of E. coli TBI(pPBGI) cells in different cellular compartments IPTG (mM)

Cytoplasm (U/L culture)

Periplasm (U/L culture)

Total (U/L culture)

Total (U/g DCW)

0.0 0.2 0.3 0.5 0.7 1.0 2.0

0.0 610.0 540.4 1050.9 1305.2 1582.9 2056.0

0.0 10.1 10.6 11.4 13.6 12.6 14.4

0.0 620.1 551.0 1062.3 1318.8 1595.5 2070.4

0.0 545.6 NA 807.0 975.1 1240.7 1657.6

DCW: dry cell weight, NA: not available.

MBP-GI of the same two cultures were compared, the increase in the concentration of the recombinant protein was only 10% for the culture induced with 1.0 mM IPTG. Regardless of IPTG concentration, the amount translocated to the periplasm did not change significantly, but the percentage of periplasmic MBP-GI to total MBP-GI appeared to drop from 1.6 to 0.7% because of the increased recombinant protein production with increased inducer concentration. The effect of IPTG concentration on cell growth was monitored as optical density vs. time in Fig. 3. IPTG addition did not immediately retard growth, cells continued to grow with the same rate until optical density reached 1.3. Cells then entered a lowered growth phase followed by the stationary phase. Retardation in cell growth seemed to follow recombinant protein expression. In the first 2 h after induction, total MBP-GI produced did not exceed 100 U (per liter culture). After the fifth hour 200 U (per liter culture) of total GI activity was measured. Maximum MBP-GI translocation to the periplasm, which was 11.4 U/L culture with 0.5 mM IPTG 4.5 4.0 3.5

OD600nm

3.0 2.5 2.0 1.5 1.0

induction, was detected at around the tenth hour after induction. The cell growth patterns observed with recombinant protein expression seems to suggest that there is toxicity associated with expression of the fusion protein. However an investigation related to the cause of toxicity was beyond the scope of this work. There are numerous studies about causes of growth-rate depression in recombinant cells, available in literature [19]. 3.3. Effect of temperature down-shift after induction on GI translocation MBP is a periplasmic protein that uses the Sec pathway of E. coli for transport. The selectivity of the Sec transport machinery for its ligands has been reported to be largely affected by a kinetic partitioning between folding of the polypeptide and recognition by the pathway chaperones [20,21]. Diamond and Randall [21] have shown that kinetic partitioning can be forced to favour association with Sec chaperones by lowering the rate of folding of the protein. Following Arhenius’s Law, reduction in temperature could decelerate folding by decreasing the rate constant of that process. This would enable Sec machinery to recognise its substrate to take it to the translocation pathway before the protein gains its final structure. In an attempt to enhance translocation of MBP-GI to the periplasm, the effect of expression of MBP-GI at lower temperatures was investigated. Recombinant cells induced with 0.7 mM IPTG at late exponential phase were assayed for GI activity in the two compartments; the cytoplasm, and the periplasm. The results in Table 3 showed that the down shift of growth temperature from 37 to 30 ◦ C or 23 ◦ C after induction in-

0.5 0.0 0

5

10

15

20

25

30

Time (hrs) Fig. 3. Effect of IPTG concentration on growth of E. coli TB1(pBGI) cells, arrow indicates the time of induction. Uninduced cells (䊊), cells induced with 0.2 mM IPTG (䊏), cells induced with 0.5 mM IPTG (), cells induced with 0.7 mM IPTG (䊉), cells induced with 1.0 mM IPTG (䊐), cells induced with 2.0 mM IPTG (䉱).

Table 3 Enzyme activity in different cellular compartments of TB1(pBGI) cells with a down-shift of temperature after induction with 0.7 mM IPTG Temperature after induction (◦ C)

Cytoplasm (U/L culture)

Periplasm (U/L culture)

Total (U/L culture)

37 30 23

1305.2 1660.1 1634.3

10.4 18.7 19.0

1315.6 1678.8 1653.3

B. Sarıyar et al. / Enzyme and Microbial Technology 35 (2004) 105–112

creased periplasmic fusion protein content from 10.4 to 18.7 and 19.0 U/L culture, respectively. The shift of temperature to 30 and 23 ◦ C increased the total active enzyme by about 25% and almost doubled periplasmic MBP-GI, although this was still only 1.5% of the total enzyme produced. 3.4. Effect of amino acids on GI translocation The two main driving forces that led us to study the effects of amino acids on the stimulation of protein expression and translocation were published observations related to the cellular stringent response [22] and weakening of bacterial cell wall [23]. Stringent response may be stimulated in the host upon depletion of specific amino acid pools used for the synthesis of heterologous proteins with amino acid compositions quite different from the average E. coli protein. Therefore addition of precursors considering the primary amino acid composition of the desired recombinant protein may enhance product yields [24]. One of the primary constituents of the cell wall peptidoglycan is alanine, found in the peptide units. It has been reported that supplements of glycine interfered with the biosynthesis of the cell wall by acting as a structural analogue of alanine [25,26], weakening the cell wall. Based on the reported results with glycine [25,27] and the most abundant amino acids found in the GI sequence [6], stimulation of synthesis and secretion of MBP-GI was studied with glycine, alanine, glutamate, leucine and threonine supplements. Since there was no detectable expression and only partial growth in chemically defined and minimal (M9, M63) media, LB was taken. However amino acid concentrations well above the initial amounts present in LB were used [28]. The results for the TB1 strain, given in Table 4, indicated that alanine and glycine caused extracellular translocation of MBP-GI. There was 24% more total GI activity with 0.5% alanine supplement. Of this, 18.5% was detected in the culture medium. With 1% alanine, total GI activity dropped by 40% but 37% of the total activity was found in the medium. These results suggested that high alanine concentrations caused leaky cell wall. Increasing glycine concentrations enhanced MBP-GI release. The high percentage Table 4 Enzyme activity in different cellular compartments of TB1(pBGI) cells with amino acid supplements, after induction with 0.5 mM IPTG Amino acid added

Whole cell extract (U/L culture)

Medium (U/L culture)

Total (U/L culture)

None Alanine (0.5%) Alanine (1.0%) Glycine (0.5%) Glycine (1.0%) Glutamate (10 mM) Leucine (0.5%)

1062.3 1077.2 402.4 530.0 85.6 577.4 188.9

0.0 244.4 233.9 365.3 131.5 0.0 11.9

1062.3 1321.6 636.3 895.3 217.1 577.4 200.8

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of GI activity detected in the medium should be from cell lysis, confirmed by the reduction in the number of viable cells. Expression and translocation results obtained with leucine, glutamate and threonine were not significant. Glycine was proposed to cause leaky cell wall, consequently periplasmic proteins are released. Supplementary experiments were conducted to check if GI activity detected in the medium had a periplasmic origin. For this reason, expression from pPBGI, which expresses cytoplasmic GI only, under glycine addition was studied. The results showed that (Table 5) at lower glycine concentrations there was no GI release. However as glycine increased, GI was detected in the medium which should be from cell lysis. Fig. 4A and B show the effect of glycine and alanine addition on cell growth. As alanine concentration increased, retardation phase attenuated but the growth rate of the exponential phase was the same regardless of alanine amount. However glycine caused lysis after induction. The higher the glycine concentration was, the sooner the cells died. During incubation, pH was monitored and the results are shown in Table 6. After induction, pH gradually increased and was around 7.5 after 30 h (extrapolation done) except in the alanine supplemented culture where pH was around 8.5. Since the highest protein expression and translocation was obtained in this culture, the result raised the question of whether external pH was an important factor affecting the expression and translocation of MBP-GI. 3.5. Effect of external pH on GI translocation Starting from the fact that external pH of cultures with alanine we around 8.5, was wanted to investigate if a basic environment could affect translocation of MBP-GI. For this reason, NaOH was added to the culture at every 3 h to keep pH at 8.0. The results showed that pH change alone enhanced neither expression nor translocation of the fusion product, therefore pH increase in alanine supplemented cultures was not the sole reason in the increase of protein release. It has been reported that low pH increased the activity of different ATPases in vitro [29,30]. Since SecA is an ATPase of the Sec pathway, actively transporting preprotein chain across the cytoplasmic membrane, we wanted to explore the possibility of increasing translocation by increasing the ac-

Table 5 Enzyme activity in different cellular compartments of DH5␣(pPBGI) cells with glycine supplements, after induction with 0.5 mM IPTG Glycine added (%)

Whole cell extract (U/L culture)

Medium (U/L culture)

Total (U/L culture)

None 0.3 0.5 0.7 1.0

156 305 231 107 48

0 0 5 42 28

156 305 236 149 76

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B. Sarıyar et al. / Enzyme and Microbial Technology 35 (2004) 105–112

Table 6 pH of TB1(pBGI) cultures with amino acid supplements Time after induction (h)

Control

Alanine 0.5%

Alanine 1.0%

Glycine 0.5%

Glycine 1.0%

Leucine 0.5%

Glutamate 10 mM

0.0 4.0 5.0 6.0 9.0 10.5 11.0 15.0 16.0 17.5 18.0 20.0 21.0 24.5 29.0

6.42

6.57

6.58

6.45

6.42

6.46 6.51

5.61

6.44

6.89

6.73 6.75

6.63

7.00

7.00

6.76

7.16

6.20

7.33 6.84 6.69

7.06

7.72

7.56 7.09

7.21

7.27 6.89

7.28

7.95

8.06

7.24 7.31 7.34

7.78

8.52

tivity of SecA. However, since cells will act to keep homeostatis, external pH changes affect intracellular pH by only 1–2 tenths of the magnitude of the imposed external pH. To investigate the effect of low pH, the growth medium was supplemented with acetic acid. However there was no significant cell growth and protein accumulation.

1.6

OD600nm

7.54

8.49

(A) 2.0

1.2

0.8

4. Conclusions

0.4

Thermostable GI from T. thermophilus was successfully cloned in E. coli using the MBP fusion system and expressed as an MBP-GI fusion protein. The product was distributed between the cytoplasm and periplasm of the bacteria. Despite of the fact that GI was produced as a fusion, it showed full activity as other proteins fused to MBP [18,31,32,33]. This proved that MBP-GI had two separate domains. The low concentration of MBP-GI measured in the periplasmic space of TB1 cells may be explained by the fast folding rate of the inherently cytoplasmic GI enzyme relative to proteins destined to be translocated through the Sec pathway. MBP-GI could not stay in a translocation competent state and folded in the cytoplasm, thus was not recognised by the secretion machinery [20,21,34]. It was also possible that processing of the fusion partners prior to translocation reduced the efficiency of translocation. Growth of recombinant TB1 cells was not retarded immediately after induction, but was severely retarded when GI expression commenced. Increasing inducer concentration increased total GI production but did not improve translocation to the periplasm. However down-shift of temperature enhanced periplasmic translocation slightly. This may be explained by reduction of folding rate of proteins at lower temperatures [21,35] and thus recognition of the protein by the translocation pathway components [36]. The most abundant amino acid in the GI sequence, alanine, increased expression of MBP-GI and enhanced its re-

0.0 0

2

4

6

8

10

12

14

Time (hrs)

(B) 2.0

1.6

OD 600 nm

7.34 7.34

1.2

0.8

0.4

0.0 0

2

4

6

8

10

12

14

16

Time (hrs)

Fig. 4. Effect of amino acid supplement on growth of induced E. coli TB1(pBGI) cells, arrow indicates the time of induction: (A) effect of glycine, 0.0% (䊊), 0.5% (䊏), 0.7% (䉱), 1.0% (䊉) and (B) effect of alanine, 0.0% (䊊), 0.5% (䊏), 1.0% (䉱).

B. Sarıyar et al. / Enzyme and Microbial Technology 35 (2004) 105–112

lease to the culture medium when supplied 0.5% in the growth medium. However increasing concentrations of alanine retarded cell growth and caused leaky cell wall. The external pH of the cultures with alanine was unusually high, however adjusting the external pH alone by adding basic solutions did not give the same results. Addition of glycine to growth media has been found to mediate expression and/or release of periplasmic proteins [24,25,37,38]. Kaderbhai [25] showed that glycine supplements could stimulate the production and discharge of the periplasmic recombinant proteins but progressive increase of glycine in the medium inhibited cell growth. Aristidou et al. [37] showed that the addition of glycine to the growth media up to 1% enhanced the release of periplasmic proteins from the cell to the broth, and a supplement below 0.7% glycine did not cause significant cell lysis. However in this study, glycine retarded cell growth and caused lysis after induction. The details of the mechanism by which addition of amino acids alter mechanism for expression and release remain to be elucidated. Many different fusion protein and tag systems can be used for translocation of proteins from the cytoplasm of E. coli. However these studies are not always successful. Secretion efficiency varies considerably depending on the characteristics of the proteins to be secreted as well as the system used.

Acknowledgments This work was supported by Bo˘gaziçi University Research Fund Projects 93A0570 and 00A503D and TÜBI˙ TAK project (TBGAG-85-DPT).

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