Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm

Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm Carol French,*’

Eli Keshavarz-Moore,+

and John M. Ward*

*Department of Biochemistry and Molecular Biology, fThe BBSRC Advanced Centre for Biochemical Engineering, Department of Chemical and Biochemical Engineering, University College London, London, United Kingdom

Numerous recombinant proteins of industrial and pharmaceutical importance are secreted to the periplasmic space of Escherichia coli; yet, very few generic periplasmic recovery methods with the potential for scaleup exist. This work describes the development of an enzymatic method for the release of recombinant proteins from the periplasm of E. coli. The simple two-step method involving resuspension of the cells in a fractionation bujher. followed by recovery of the periplasmic fraction, has been shown to be feasible for use in both the laboratory and 5-l scale. For the eficient release of a recombinant Streptomyces thermoviolaceus cY-amylase from the E. coli periplasm, it has been shown that an osmotic shock must immediately follow lysozyme treatment to obtain high yields. The phase of growth and level of recombinant protein expression has an affect on the recovery from the E. coli periplasm. Yields of 70-90% of recombinant u-amylase were recovered from the periplasm of stationary phase E. coli cells in both laboratory and 5-l scale experiments. Keywords:

Periplasmic

release; recombinant

protein; Escherichia cob; a-amylase

Introduction Escherichia coli has been widely used for the production of heterologous proteins in the laboratory and industry. E. coli does not generally excrete proteins to the extracellular medium apart from colicins and hemolysin.’ Heterologous proteins expressed in E. coli can be intracellular, sequestered in the cytoplasm, or secreted to the periplasm if they possess a N-terminal signal sequence. The secretion of recombinant proteins to the periplasmic space has numerous advantages over expression in the cytoplasm. The periplasmic space contains only 7 out of the 25 known cellular proteases’ and comprises only 4-8% of the total cell protein.3 The mature secreted protein does not incorporate N-formyl methionine and the oxidative environment of the periplasm facilitates correct disulfide bonding and protein folding.4 Numerous heterologous proteins have been secreted to the periplasmic space of E. coli, including antibody fragments,5 ribonuclease A,6 HIV-l receptors,’ and interleukin-2.8 Address reprint requests to Dr. John M. Ward, Dept. of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, United Kingdom Received 9 August 1995; revised 17 November 1995; accepted 13 December 1995

Enzyme and Microbial Technology 19:332-338, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 100’10

If the recovery of a recombinant protein from the periplasm can be achieved in a small volume without contamination by cytoplasmic proteins, subsequent purification steps are greatly simplified. There are numerous small-scale periplasmic release methods in the literature, but these do not translate easily and efficiently to a large-scale process. Treatments involving chemicals such as chloroform,9 guanidine-HCl, and Triton X-lOO1o have been used to release recombinant proteins from the E. coli periplasm; however, these chemicals are not inert and may have detrimental effects on many recombinant protein products or subsequent purification procedures. Glycine treatment of E. coli cells, causing permeabilization of the outer membrane, has also been reported to release the periplasmic contents”; however, these treatments have been designed for specific systems and are generally unsuitable as large-scale methods. The most widely used methods of periplasmic release are osmotic shock’2.‘3 and lysozyme/EDTA treatment.‘“.” Many different modifications of these methods have been used on a wide range of expression systems with varying degrees of success. i6,17 Although these methods work efficiently on a laboratory scale, they involve too many steps for an efficient large-scale recovery process. In this paper, we report the development and optimiza-

0141-0229/96/$15.00 PII SO141-0229(96)00003-7

Development

of a simple

method

tion of an enzymatic release method to recover recombinant periplasmic proteins which is suitable for large-scale use. The effects of overexpression of the recombinant protein, S. thermoviolaceus ol-amylase, and the growth phase of the host organism on the recovery are also discussed.

Materials

and methods

Strains and plasmids The E. coli K12 bacterial strain JMl07 (e&Al, gyrA96, fhi, hsdR17, supE44. relA1, A, A(&-proAB), [F’, traD36, proAB, la@, ZAM15])‘8 was used in this study. The plasmids used were pBGS 19- I’) and ~QR300.~~ The construction and use of the plasmid pQR126 is described in the text.

DNA manipulations All restriction endonuclease digests, ligations, and transformations were performed according to Sambrook et ~1.~’ Restriction enzymes were obtained from New England Biolabs (Beverly, MA, U.S.).

Growth conditions Strains were maintained on nutrient agar plates supplemented with 1% (w/v) potato starch (Sigma S-4251, St. Louis, MO, U.S.) and 20 pg ml-’ kanamycin (Sigma K-4000) or 100 p,g ml-’ ampicillin (Sigma A-9518). Cultures were grown in nutrient broth (Oxoid No. 2, Basingstoke, Hants, UK) or Terrific Broth (TB)22 supplemented with 10 mg ml-’ kanamycin or 100 mg ml-’ ampicillin to maintain plasmid stability. Starter cultures were prepared by inoculating 200 ml sterile medium with 2-3 colonies from a fresh agar plate and incubated overnight (17 h) in an orbital shaker (200 rpm) at 37°C. Shake-flask cultures (200 ml) were inoculated with a 4% (v/v) starter culture and incubated in an orbital shaker (200 rpm) at 37°C. Batch fermentations were camied out in a 7 I stirred tank (5 1 working volume) LH2000 bioreactor. Cultures were inoculated with a 4% (v/v) starter culture and grown at 37°C with an aeration rate of 0.5 vvm and an agitator speed of 1000 ‘pm. Dissolved oxygen tension and pH were monitored by Ingold electrodes. The gas exhaust compositions, monitored by on-line mass spectroscopy, and the cultivation parameters were logged using BioI software (Biotechnology Computer Systems, London, UK). Cell

fractionation

Samples (5 ml) were harvested by centrifugation at 13,000 g and the supematant, referred to as the extracellular fraction, was carefully removed. The cell pellet was washed in 50 mu Tris-HCI pH 7.5 (Sigma T-1503) and recentrifuged. The periplasmic contents were then released by various methods as described. Osmotic shock. Osmotic shock treatment was a modification of the method by Nossal and Heppel.12 The cell pellet was resuspended in a buffer (1 ml) containing 20% sucrose and various concentrations of Tris-HCl pH 7.5 and Na,EDTA (Fisons, Loughborough, Leicestershire, UK). After a static incubation at room temperature for 15 min, the osmotically fragile cells were harvested by centrifugation at 13,000 g for 15 min. The supematant was removed and the cell pellet resuspended in 1 ml distilled H,O (4°C) and incubated with gentle agitation at 4°C for 15 min. The resulting spheroplasts were harvested (13,000 g at 4°C) and the supematant. referred to as the periplasmic fraction, was recovered. Chloroform treatment. The periplasmic using the method of Ames ef aL9

contents

were released

for the recovery

of recombinant:

Glycine treatment. The periplasmic the method of Ariga et al.”

C. French et al.

contents were released using

LysozymeIEDTA treatment. LysozymelEDTA treatment was a modification of the methods of Neu and Heppel14 and Witholt et al. I5 The cell pellet was resuspended in a buffer ( I ml) containing 20% sucrose and various concentrations of lysozyme (E.C. 3.2.1.17) (Sigma L-6876, chicken egg white), Tris-HCI pH 7.5. and Na,EDTA. After a static incubation at room temperature for 15 min, the cells were harvested by centrifugation at 13,000 g for 15 min and the supematant was recovered as the periplasmic fraction. The remaining cell pellet was washed in 1 ml dH,O at 4°C and harvested (13,000 g at 4°C). The supematant was recovered and designated as the “cold water wash fraction.” Following each of these periplasmic fractiqnation methods. the cell contents, referred to as the cytoplasmic fraction. were prepared by sonication (6 x 10 s bursts with IO s intervals) and centrifugation ( 11,500 g).

Large-scale

cell fractionation

A 5-l culture was harvested in a Sharples 1P continuous centrifuge (Pennwalt Ltd, Camberley, Surrey, UK) at 45,000 rpm. The cell paste from 5 1 of culture was then resuspended using a Silverson homogenizer in 1 1 of fractionation buffer containing 500 p,g ml-’ lysozyme. 20% sucrose. and 1 mM EDTA. This was incubated for 15 min at room temperature before an equal volume of water was added. The suspension was then incubated for a further 15 min at room temperature. The periplasmic fraction was then recovered in a Sharples 1P centrifuge. Control samples of 1 ml volume were taken after the water dilution step and were either harvested in an Eppendorf microfuge or filtered through a 0.2 pm membrane to compare the recovery of the periplasmic fraction on a small and large scale.

cr-amylase assa_v a-Amylase activity was determined by measuring the rate of digestion of starch using an I2 (Fisons) complex.2’ Enzyme samples were diluted into 1 ml of freshly prepared 0.25% (w/v) soluble starch (Sigma S-2630) in 15 mM sodium phasphate buffer pH 5.8 which had been previously heated to boiling point and filtered while hot (Whatman No. 1 filter paper). The enzyme and starch reaction mixture was incubated at 50°C; 50 p,l aliquots were removed at various time points and diluted into 1 ml iodine solution [freshly prepared by adding 200 ml 2.2% I,/4.4% KI (w/v, Sigma P-4286) stock solution to 100 ml of 2% (w/v) KI solution]. The rate of decrease in absorbance at 620 nm was measured.

Protein analysis Protein concentration was determined by a calorimetric assay USing BioRad Protein assay reagent (Richmond, VA).14 Bovine serum albumin was used as the standard.

Results Plasmid construction The a-amylase gene (amy) encodes a protein of 460 amino acid residues with a molecular weight of 47 kDa following cleavage of a 28 amino-acid residue sigma1 peptide. A 3.4 kb HindIIUPstI fragment encoding the S. themviolaceus amy gene was excised from pQR300 and cloned into HindIII/PstI digested pBGS19- to form the construct pQR126 (Figure I). This plasmid construction removed a 150 bp region of DNA upstream of the amy gene which encoded Enzyme Microb. Technol.,

1996, vol. 19, October

333

Papers B

P

S

HB PlacZ

*my t c---

5.4kb

PQM~ (7.07 kb)

___)

P

WC

S

H

18)

PklcZ

_amy +

Figure 1

3.4 kb

A 3.4 kb Hindllllfsd

-

fragment

(pBGS 19-j

containing

the S. ther-

moviolaceus cu-amylase gene was excised from pQR3003’ and cloned into pBGSlS- to form the construct pQR126. The arrows are diagrammatic and show the direction of transcription from the /acZ promoter and the orientation of the cY-amylase gene. BarnHI, B; HindIll, H; Pstl, P; and Sphl, S

the nature a-amylase promoter.25 The reduced distance between the 1ucZ promoter of the vector and the translational start site of the c+amylase gene in pQR126 increased the production of a-amylase by fivefold (5 U ml-’ culture) compared to the original construct pQR300 (1 U ml-‘, data not shown).

Small-scale

development

of periplasmic

release

Periplasmic release methods were initially compared on a small scale from the stationary phase (12 h) 200 ml nutrientbroth shake-flask cultures. The same cell batch of JM107 pQR126 was used for all of the following periplasmic release methods (see MATERIALSAND METHODS).The periplasmic fraction was assayed for a-amylase and protein content to measure the efficiency of each periplasmic release method. The remaining cell debris was then sonicated to obtain the cytoplasmic fraction. This was assayed for ol-amylase and protein content to monitor the level of periplasmic ol-amylase that remained cell associated after each release method. Figure 2 shows the cellular distribution of ol-amylase from a stationary phase culture of JM107 pQR126 following osmotic shock, chloroform, glycine, and lysozyme/ EDTA treatments. The total cellular a-amylase was calculated from the sum of the periplasmic and cytoplasmic fractions and, where applicable, the cold water wash fraction. The total level of a-amylase released from the periplasm by the osmotic shock and the chloroform treatments was lower than that released by disrupting the same volume of cells by sonication. This implied that these methods did not effciently disrupt the cell to release the total cellular a-amylase. An increase in Tris-HCl concentration from 50 mM to 200 mu in the osmotic shock procedure improved the release of periplasmic Lw-amylase from 1.7 U ml-’ culture to 2.8 U ml-’ culture, yet this represented only 53% of the total cellular a--amylase. Glycine treatment,’ l where harvested cells are resuspended in a 1% glycine solution, released only 1 U ml-’ culture of a-amylase from the periplasm (Figure 24. This represented only 17% of the total cellular a-amylase. Lysozyme/EDTA treatment greatly increased the yield of a-amylase released from the periplasm compared to the 334

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1996, vol. 19, October

Figure 2 Cellular distribution of a-amylase following various periplasmic release methods on overnight (18 h) stationary phase JM107 pQR126 nutrient-broth cultures (see MATERIALS AND METHODS). Sonication control, a; osmotic shock, b; chloroform treatment, c; glycine treatment, d; lysozyme/EDTA treatment, e; combined lysozyme/osmotic shock treatment, f. Data shown in the Figure were from one set of experiments. Similar results were obtained in four separate experiments. Periplasmic fraction, 0; cytoplasmic fraction, n ; and cold water wash fraction, K

other release methods (Figure 2e). Lysozyme cleaves the periplasmic cross-linked peptidoglycan gel matrix.‘6 The requirement for lysozyme to release the a-amylase from the periplasm implied that the recombinant protein was entrapped within or behind the peptidoglycan gel matrix. The maximum level of ol-amylase released into the periplasmic and “cold water wash” fractions was almost twofold greater (10.8 U ml-’ culture) than the amount of enzyme released by sonication of the cells (5.9 U ml-’ culture); however, sonication may not have released the total cellular a-amylase if the protein is entrapped by the peptidoglycan gel matrix. No loss of activity of a--amylase caused by sonication could be detected. The proposed entrapment of the ol-amylase within the peptidoglycan gel explains why the previous methods lacking lysozyme, osmotic shock, glytine, and chloroform treatments did not release the total periplasmic a-amylase. Increased concentrations of Tri-HCl and EDTA in the lysozyme fractionation buffer released greater levels of a-amylase from the periplasm. This was presumably due to

Development

of a simple

method

for the recovery

of recombinant:

C. French et al.

a greater destabilization effect on the outer membrane facilitating release from the periplasm; however, the lysozyme/EDTA treatment did not fully release the total periplasmic a-amylase in a single fraction since 3 l-42% of the ar-amylase was released by the “cold water wash” step. This procedure would be unsuitable for a large-scale process since it involves multiple centrifugations to recover the total cellular cl-amylase.

Combined lysozyme/osmotic

shock treatment

The above results demonstrated that both lysozyme/EDTA treatment and osmotic shock were required to release maximum levels of the periplasmic c-w-amylase. In order to release a-amylase in a single fraction, a combined lysozyme/ osmotic shock treatment was developed. Harvested cells were resuspended in a fractionation buffer (20% of the original culture volume) containing 500 pg ml-’ lysozyme, 20% sucrose, and varying concentrations of EDTA and Tris-HCl. The cell suspension was incubated statically for 15 min at room temperature before an equal volume of cold water was added. The suspension was incubated for a further 15 min before recovery of the periplasmic fraction by centrifugation. This combined treatment released a maximum level of 90% (11.8 U ml-‘) of the total cellular o-amylase in a single periplasmic fraction in the presence of 1 mM EDTA and 200 mM Tris-HCl (Figure 2j). Further optimization experiments on the combined lysozyme/osmotic shock treatment were carried out. For the maximal release of periplasmic ol-amylase, a lysozyme concentration of 500 p,g ml-i or above was required. No further u-amylase was released from the periplasm by increasing the EDTA concentration above 1 mu (data not shown). Nossal and Heppel’? similarly found that increasing the concentration of EDTA in an osmotic shock procedure had little effect upon the release of periplasmic proteins.

Effect of growth and overexpression periplasmic recovery

on

Figure 3 shows the cellular distribution of cY-amylase throughout growth of JM107 pQR126 in nutrient-broth shake flasks. The combined lysozyme/osmotic shock method efficiently released 80-100% of the periplasmic cl-amylase in a single periplasmic fraction at all stages of growth. o-Amylase was recovered at a yield of 16 U ml-’ culture from JM107 pQR126 after 24 h of growth which was equivalent to 22% of the recovered periplasmic protein (as determined by SDS-PAGE). When JM107 pQR126 was cultured in Terrific Broth which gave a threefold increase in o-amylase production (45 U ml-’ culture), it became increasingly difficult during the release procedure to harvest spheroplasts of late exponential and stationary phase samples. This was attributed to an increase in viscosity caused by the partial release of nucleic acids and made the recovery of the periplasmic fraction virtually impossible. Complete cell lysis had not occurred since the majority of the intracellular protein (66%) was recovered in the cytoplasmic fraction after sonication; however, some cytoplasmic protein was released into the

TIME (H) Figure 3 Time course of JM107 pQR126 cultured in nutrientbroth shake flasks showing the total cy-aqylase distribution. Cells were fractionated using the combined’ lysozyme/osmotic shock treatment (fractionation buffer: 20% sucrose, 200 f?‘?M Tris-WI 1 mM EDTA, 500 pg ml-’ lysozyme). Data shown in the Figure were from one set of experiments. Similar results were obtained in three separate experiments. Extracellular fraction, cytoplasmic fraction, -O-; -D-; periplasmic fraction, -0-; and OD 600 nm, -Cl-

periplasmic fraction, lowering the specific activity of the recovered recombinant cY-amylase. This “partial cell lysis” during periplasmic release occurred at the same period of growth as leakage of a substantial amount of periplasmic cx-amylase to the extracellular medium. Leakage of recombinant proteins from the periplasm is common,27.28 typically resulting from an increase in outer membrane permeability caused by high-level recombinant protein expression. It was discovered that the presence of high concentrations of Tris-HCI (e.g., 200 mM) caused the “partial cell lysis” of JM107 pQR126, leading to the increased viscosity of the periplasmic fraction. Early stationary-phase Terrific Broth shake-flask cultures could only be pelleted without any increase in viscosity when fractionated in the presence of 10 mM Tris-HCl or in the absence of Tris-HCl (Table I). Periplasmic release using 10 mu Tris-HCl or in the absence of Tris-HCl affected the yield with only 70-82% of the total o-amylase and 54-67% of the periplasmic protein being recovered compared to the 200 mM Tris-HCl extraction. Spheroplasts from Terrific Broth bioreactor cultures obtained using the standard 1 ml culture samples could only be harvested without any increase in viscosity in the absence of Tris-HCl. This resulted in a 41% reduction in the recovery of a-amylase compared to the 200 mM Tris-HCl extraction; however, the specific activity of the recovered periplasmic c-u-amylase increased by almost twofold due to the lower level of protein released when the cells were fractionated in the absence of Tris-HCl (Table I). This reduced level of contaminating protein in the periplasmic recovery fraction will simplify any subsequent purification protocols. This data demonstrates that fermentor culture samples are more sensitive to Tris-HCl than cells cultured in shake flasks. This implies that the outer membranes of cells cultured in the fermentor are weaker than those of shake-flask cultures Enzyme Microb. Technol.,

1996, vol. 19, October

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Papers Table 1

Effect of Tris-HCI

on periplasmic

release

Shake-flask culture Tris-HCI

(mM)

Ability to pellet ol-Amylase (U ml-’ culture) Protein (pg ml-’ culture) % cr-Amylase recovery’ % Protein recoverya Specific activity (U mg-’ protein)

Fermentation

culture

200

10

0

200

10

0

30.1 281 100 100 107

2+4.7 190 82 68 130

2+.1 153 70 54 138

41.6 1,008 100 100 41

26 736 63 73 35

2+4.5 320 59 32 76.6

Comparative levels of cY-amylase and protein released in the periplasmic fraction by the combined lysozyme/osmotic shock method using different Tris-HCI concentrations. Experiments were performed on JM107 pQR126 Terrific Broth stationary phase (12 h) shake flask and fermentation samples. Pelleting of spheroplasts, +; inability to pellet, -. Data shown in the Table were from a single set of experiments. Similar results were obtained in three separate experiments Vecovery percentages were calculated using the 200 mM extraction values as 100% recovery

which could have been caused by shear stress in the fermentor vessel. The increase in protein in the fermentor cultures and the weakening of the outer membrane could also be attributed to the faster cell growth and higher cell density in the fermentor. The cellular distribution of recombinant o-amylase in a selective JM107 pQR126 5-l batch Terrific Broth fermentation was monitored for 24 h on a laboratory scale using the standard 1 ml culture samples (Figure 4). Tris-HCl was omitted from the lysozyme/osmotic shock release treatment to prevent “partial cell lysis” occurring. The combined lysozyme/osmotic shock treatment recovered 75-80% of the total cellular o-amylase from the periplasm of stationary phase cultures; however, only 55-72% of the total cel-

20

25 15

1

10

8

5

0 5

10

15

20

lular a-amylase was recovered from exponential phase cultures. This implies that the outer membrane of the recombinant cells is more sensitive to the lysozyme/EDTA treatment during the stationary phase than the exponential phase. This can be attributed to the high level recombinant protein expression and accumulation of a-amylase in the periplasm leading to leakage through the outer membrane. This is in contrast to previous data which showed that nonrecombinant E. coli cells are more resistant to osmotic shock during the stationary phase.12,29

Processing of a j-liter culture A 5-l Terrific Broth JM107 pQR126 fermentation culture was harvested after 24 h of growth (see MATERIALS AND METHODS) to give a biomass of 24 g 1-l wet weight. Largescale periplasmic release using the combined lysozyme/ osmotic shock methodology recovered 73% of the total cellular o-amylase (12.92 U ml-‘) compared to the standard laboratory scale (1 ml) experiment where 100% was recovered (Table 2). The protein levels released by centrifugation of the spheroplasts in the Eppendorf microfuge (222.1 kg ml-‘) were three- and fourfold greater compared to the same spheroplasts being filtered (56 p,g ml-‘) or harvested in the Sharples IP centrifuge (76 pg ml-‘). This data shows that negligible damage to the osmotically fragile spheroplasts was incurred by harvesting in the Sharples 1P centrifuge. Recovery of the periplasmic fraction in the Sharples 1P centrifuge or by filtering increased the specific activity of the recovered a-amylase by three- to fivefold compared to samples recovered in the microfuge.

25

TIME ‘30

Discussion Figure 4 Time course of JM107 pQR126 cultured in Terrific Broth batch fermentation cultures showing the total a-amylase cellular distribution. Cells were fractionated using the combined lysozyme/osmotic shock treatment without any Tris-HCI present to ensure spheroplast pelleting (fractionation buffer: 20% sucrose, 1 mM EDTA, 500 pg ml-’ lysozyme). Data shown in the Figure were from one set of experiments. Similar results were obtained in four separate experiments. Extracellular fraction, -W-; periplasmic fraction, -0-; cytoplasmic fraction, -O-; and OD, 600 nm, --O-

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Osmotic shock alone, as used by Nossal and Heppel,‘” did not efficiently release the S. themzovioluceus o-amylase from the periplasm of E. cd. The addition of lysozyme to degrade the peptidoglycan matrix was required for efficient release, implying that the recombinant protein may have been entrapped within the peptidoglycan gel matrix. This may explain the poor o-amylase recovery obtained by sonication and osmotic shock, and glycine and chloroform treat-

Development Table 2 Comparison mic release

Spheroplast recoven/ method Sharples 1 P centrifuge Eppendorf microfugeb 0.2 pm filterb Standard laboratory methods (1 ml)

between

of a simple method

small- and large-scale

a-Amylase (U ml-’ culture)

Protein (pg ml-’ culture)

12.92 (73%)”

76

(33%)

12.35 (69%) 15.2 (85%)

222.1 (97%) 56 (25%)

17.8

228

periplas-

Specific activity (U mg-’ protein)

170

(218%)

55.6 (71%) 271 (347%)

78

Comparison of laboratory and pilot-plant scale periplasmic release using the combined lysozyme/osmotic shock treatment (fractionation buffer 20% sucrose, 1 mM EDTA, 500 pg ml-’ lysozyme) on JM107 pQR126 stationary phase (24 h) fermentation cultures. Samples were fractionated on the standard laboratory scale (I ml) and on a large scale (5 IL Spheroplasts were pelleted in an Eppendorf microfuge (small scale) or a Sharples IP continuous centrifuge (large scale). Data shown in the Table were from a single set of experiments. Similar results were obtained in three separate experiments aFigures in parentheses denote the recovery percentages and were calculated using the standard laboratory method values as 100% recovery bControl samples of 1 ml volume were taken from the largescale release experiment before the bulk of the spheroplasts were harvested to recover the periplasmic fraction. The periplasmic fraction in the control samples was then recovered by harvesting in a microfuge (13,000 g for 10 min) or filtering through a 0.2 pm filter

ments. Carter et al. ” also found that simple osmotic shock was not sufficient to release recombinant Fab’ fragments from the periplasm of E. coli; the use of lysozyme was necessary. The maximum ol-amylase recovery was achieved when a water dilution step followed the lysozyme/EDTA treatment. The addition of an equal volume of water causes a small osmotic shock, thereby diluting the periplasmic contents by twofold. Witholt et all5 stated that if the polysaccharide chains of the peptidoglycan gel were stacked as closely as predicted by Formanek et al,30 lysozyme would be unable to bind to the matrix. A sudden influx of water would cause the cells to swell and the periplasmic space would be enlarged. This would increase the distance between the polysaccharide chains and potentially enhance the binding of lysozyme to the matrix. Similarly, Birdsell and CotaRobles3’ found that following plasmolysis in the presence of lysozyme, Tris-HCl, and sucrose, spherical spheroplasts formed only on the addition of EDTA or a sudden water dilution. The removal of Tris-HCl from the fractionation buffer resulted in a loss of up to 40% in the recovery of r;u-amylase; however, this loss is compensated by the additional reduction in total protein levels released which led to a twofold increase in a+amylase specific activity. This higher specific activity will aid further purification of recombinant cY-amylase since the total contaminating protein has been reduced by threefold.

for the recovery of recombinant:

C. French et al.

The 5-l recovery experiment has demonstrated that it is feasible to carry out the periplasmic recovery method using pilot plant machinery such as the Sharples 1P continuous centrifuge and Silverson homogenizers. The scaling up of the process to larger volumes using comparable pieces of equipment should result in the same high recovery of periplasmic ol-amylase. This experiment also showed that the method of spheroplast recovery by microfuge, Sharples lP, or filtering can affect the levels of total protein recovered by up to fourfold. The lower levels: of contaminating protein released using the Sharples 1P and by filtering will aid any future downstream purification steps.

Conclusions The recovery of a recombinant protein from the periplasm of E. coli cells using the combined lysozyme/osmotic shock method is appropriate as a rapid small-scale analytical method and for a larger scale process. After the cells have been harvested, the method involves only a single centrifugation step which could be easily replaced by filtration. The recombinant product is recovered in only 40% of the original culture volume where the ratio of product to contaminating intracellular protein is high. In 5-l culture trials, the treatment recovered up to 73% of active periplasmic cY-amylase from the E. coli cells, comparing favorably to small-scale methods.‘3.17.”

Acknowledgments UCL is the Interdisciplinary Research Centre for Biochemical Engineering of the BBSRC. The support of the Council to the participating UCL departments is gratefully acknowledged. We are grateful to Dr. D. A. Cowan for his assistance in the preparation of this manuscript.

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Halfmann, G., Brailly, H., Bemadac, A.. Montero-Julian, F. A., Lazdunski. C., and Baty, D. Targeting of interleukin-2 to the periplasm of Escherichia coli. 3. Gem Microbial. 1993, 139, 2465-2473 Ames, G. F.-L., Prody, C., and Kustu, S. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J. Bacreriol. 1984, 160, 1181-1183 Naglak, T. J. and Wang, H. Y. Recovery of a foreign protein from the periplasm of Escherichiu coli by chemical permeabilisation. Enytze

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