Scholarly article on topic 'Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli'

Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Giovana C. Barazzone, Rimenys Carvalho, Stefanie Kraschowetz, Antonio L. Horta, Cíntia R. Sargo, et al.

Abstract New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surface proteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensable for virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of protein sequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in 99% of strains associated with pneumococcal invasive disease. Hence, the purpose of this work was to develop an industrial production and purification process of His-tagged recombinant fragment of PspA in E. coli BL21 (DE3), rfPspA245 from family 1. Fed-batch cultivations in 5-L bioreactors with defined medium were carried out using glycerol as carbon source. It was obtained circa 60g/L of dry cell weight and 3.0g/L of rfPspA. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. The clarification step was done by centrifugation. The results of chromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographic sequence anion exchange (Q-Sepharose) followed by metal affinity (IMAC-Sepharose), the rfPspA245 was obtained with 67% and 97% of purity respectively for each step and final recovery of 23%. In conclusion, the purification process was developed and rfPspA245 was obtained with high purity, but the recovery should still be improved.

Academic research paper on topic "Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli"

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Vaccinology

Procedia in Vaccinology 4 (2011) 27-35

www.elsevier.com/locate/ procedia

4th Vaccine and ISV Annual Global Congress

Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli

Giovana C. Barazzonea, Rimenys Jr. Carvalhoa, Stefanie Kraschowetzb, Antonio b b b b C. L. Horta , Cíntia R. Sargo , Adilson J. Silva , Teresa C. Zangirolami , Cibelly

Goularta, Luciana C. C. Leitea, Martha M. Tanizakia, Viviane M. Gon?alvesa,

Joaquin Cabrera-Crespoa*

aCentro de Biotecnologia, Instituto Butantan, Av Vital Brazil 1500, 05503-900, Säo Paulo, Brazil bDepartamento de Engenharia Química, Universidade Federal de Säo Carlos, Rodovia Washington Luiz Km 235, 13565-905, Säo

Carlos, Brazil

Abstract

New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surface proteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensable for virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of protein sequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in 99% of strains associated with pneumococcal invasive disease. Hence, the purpose of this work was to develop an industrial production and purification process of His-tagged recombinant fragment of PspA in E. coli BL21 (DE3), rfPspA245 from family 1.

Fed-batch cultivations in 5-L bioreactors with defined medium were carried out using glycerol as carbon source. It was obtained circa 60 g/L of dry cell weight and 3.0 g/L of rfPspA. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. The clarification step was done by centrifugation. The results of chromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographic sequence anion exchange (Q-Sepharose) followed by metal affinity (IMAC-Sepharose), the rfPspA245 was obtained with 67% and 97% of purity respectively for each step and final recovery of 23%. In conclusion, the purification process was developed and rfPspA245 was obtained with high purity, but the recovery should still be improved.

© 2011 Published by Elsevier Ltd. Selection and peer-review under responsibility of Prof. Ray Spier

* Corresponding author. Tel.: 55 11 37267222; fax: 55 11 37261505 E-mail address: jcabrera-crespo@butantan.gov.br.

1877-282X © 2011 Published by Elsevier Ltd. Selection and peer-review under responsibility of Prof. Ray Spier doi:10.1016/j.provac.2011.07.005

Keywords: PspA; Streptococcus pneumoniae; production; purification

1. Introduction

Streptococcus pneumoniae is a Gram-positive microorganism with a polysaccharide capsule, causative agent of bacterial pneumonia, otitis media, meningitis and sinusitis. It is a human pathogen transmitted from person to person by aerosol.

The incidence of pneumococcal disease and mortality is higher in children under 5 years of age, in the elderly and in immunocompromised individuals [1, 2]. A mortality rate of 5 - 22% has been associated with the initial phase of the disease and even in cases with antibiotic susceptible strains; there is still a mortality of 10% in pneumonia and 30% in meningitis [3], more frequent in the first months of life [4]. This situation is worse when chemotherapy fails due to resistant strains [5].

The most important pneumococcal virulence factor is the capsular polysaccharide, whose structure defines the serotypes. In Brazil, there are 12 prevalent serotypes, which are responsible for 75% of the pneumococcal infections, and 45% of the pneumococcal infections are related to the serotypes 14, 6B and 1. The serotypes 1 and 6B are prevalent in all ages; the serotype 14 is prevalent in children and the 3 and 4 in adults [4].

Nomenclature

PspA Pneumococcal surface protein A

IMAC immobilized metal-ion affinity chromatography

kan kanamycin

HCD high cell density

OD optical density

HPLC high performance liquid chromatography

SDS sodium dodecyl sulfate

PAGE polyacrylamide gel electrophoresis

ms(t) glycerol mass flow rate (g/h)

Yx/s yield factor on biomass (g dry cell weight/g consumed glycerol)

^set desired specific growth rate during the fed-batch phase (h-1)

m maintenance coefficient (0.025 g/g.h)

Xf cell concentration at the beginning of feeding (g dry cell weight/L)

Vf medium volume (L) at the beginning of feeding

tf instant at the beginning of feeding (h)

t time

IPTG isopropyl ß-D-thiogalactopyranoside

PMSF phenylmethylsulfonyl fluoride

kDa kilo Daltons

So far all the current pneumococcal available vaccines are based on free capsular polysaccharides or polysaccharides conjugated to carrier proteins. Conjugation of the polysaccharide with carrier proteins induces T-cell dependant immunity, high antibody production and memory B-cells. The first used conjugated pneumococcal vaccine was licensed in 2000 (Prevenar, Wyeth) containing 7 serotypes (4, 6B, 9V, 14, 18C, 19F e 23F) conjugated with a mutated diphtheria toxin, CRM197. This formulation has been effective against the pneumococcal invasive disease in children lower than 2 years old [6]. The more commonly used carrier proteins are the tetanus and diphtheria toxoids [7], the genetically mutated diphtheria toxin CRM197 [8] and the outer membrane protein complex of Neisseria meningitidis [9]. Although the conjugated pneumococcal vaccines are highly considered in the invasive infection, the variability of serotypes continues to be an obstacle [10], technically limiting the number of possible antigens included in the vaccine [11].

In order to broaden the coverage of the vaccine, a new conjugate vaccine using the three most prevalent polysaccharides in Brazil, serotypes 14, 6B and 1, conjugated to PspA are being developed. The PspA was shown to induce systemic antibody response and protection against challenge with a virulent strain in mice by a mechanism of inhibition of complement deposition in the bacterial surface. Furthermore, since the PspA structure is like a long tail, there is a region which is not covered by the capsule [12]. The N-terminal region of PspA contains most of the immunogenic epitopes of this molecule [13] and is capable of protecting mice against an invasive challenge with virulent pneumococci [14]. However, this region exhibits serological variability, leading to the classification of the protein in three families. The family 1 consists of clades 1 and 2, family 2 of clades 3, 4 and 5, and family 3 of clade 6 [15]. The families 1 and 2 are present in around 99% of pneumococcal strains; therefore the N-terminal region of one representative of family 1, PspA245 was chosen to be cloned in Escherichia coli in order to develop the production process for the carrier proteins of this new conjugate pneumococcal vaccine.

E. coli is the bacterium most used in expression of heterologous protein. This system allows obtaining high-density cell cultures [16]. However, rarely, the recombinant protein is obtained in the broth culture. So, it is necessary to lyse the cells to extract the protein of interest. Due the enormous amount of impurities released by the lysis, several purification steps are necessary. After lysis, clarification and chromatographic steps are employed. The right choice of chromatographic conditions can generate the protein in high yield and purity degree [17]. Ion exchange chromatography is widely used because is simple to operate, allows a greater flow and has a lower cost. Despite of higher cost, immobilized metal-ion affinity chromatography (IMAC) is also very common due to the fact His-tagged recombinant proteins, as the fragments of PspA which were synthesized with six histidine residues, have affinity for metals like Ni+2 [18].

2. Materials and Methods

2.1. Production of rfPspA245 in E. coli BL21(DE3)

The N-terminal fragment of pspA245 gene was cloned into pET37b+ and expressed in E. coli BL21(DE3). The frozen stock was spread in agar M9 medium with kanamycin (kan). High cell density (HCD) medium [19] containing 20 g/L glycerol was used for cultivation of the inoculum and the same medium with 40 g/L glycerol was used for batch cultures in 5L-reactor BioFlo 2000 (New Brunswick). The cell concentration was measured by 0D600nm. The glycerol and acids concentrations were analyzed by HPLC (Aminex HPX-87H, BioRad) and the protein by SDS-PAGE 12%. The mass flow rate of the

carbon source during the fed-batch phase was calculated according to the equation (1) and the induction was done with 1 mM IPTG.

mS (t) =

lx / s

• ßset + m

■ XF ■ VF • eßset '(t~tp)

2.2. Purification of rfPspA245 produced in E. coli BL21(DE3)

After the cultivation, the cellular suspension was centrifuged (17,969 g) by 30 minutes at 4°C. The cell mass was frozen. The cell pellets (400g) were resuspended in 1.0 L of lysis buffer (25 mM tris pH 8.0 + 0.1% triton X-100) with a protease inhibitor, 1.0 mM PMSF. A homogeneous suspension was obtained in a mixer (CAT X520) and disrupted by a high pressure continuous homogenizer (APV-Gaulin) in a close loop for 12 minutes at 600 bar. The homogenizer has a jacketed reservoir and a tube-and-shell heat exchanger in the inlet and outlet, respectively, to control the temperature during the lysis under 12°C. Samples were taken every minute to determine the efficiency of lysis. The 100% of lysis efficiency was considered the 0D600nm of the cell suspension after treatment with 0.1M NaOH.

The homogenate clarification was done by centrifugation (17,696 g) for 2 h at 4°C. The supernatant was filtrate in a membrane (0.45 ^m) to obtain the clarified homogenate.

The chromatographic steps were done using an Akta Explorer (GE Heathcare). The flow was 50 mL/min in columns XK 50. The resins employed were Q-Sepharose Fast Flow (anion exchange) and IMAC-Sepharose (immobilized metal-ion affinity chromatography). All material was purchased from GE Healthcare.

We evaluated two chromatographic sequences: Q-Sepharose followed by IMAC-Sepharose and IMAC-Sepharose followed by Q-Sepharose. In the case of Q-Sepharose, the elution buffer was 25 mM sodium acetate pH 6.5 + 200 mM NaCl. IMAC-Sepharose was loaded with NiS04 and the elution was done with 20 mM phosphate buffer pH 7.4 + 200 mM imidazol. The conditions of binding, wash, elution and cleaning are showed in Figure 1 (Q-Sepharose) and Figure 2 (IMAC-Sepharose).

Q - Sepharose Sample

10« mM NaCl 200 mM NaCl 3(10 mM NaCl

5(H) mM NaCl

Flow Thruitgh 25 mM sodium acetate pH 6.5

Wash I

Elution

Wash 2

Clean ill!!

QFI QF2 QF3 QK4

Fig. 1. Chromatographic conditions used for purification of rfPspA245 in Q-Sepharose.

Fig. 2. Chromatographic conditions used for purification of rfPspA245 in IMAC-Sepharose.

2.3. Analytical Methods

Protein quantification was done according to Bradford [20], using Bradford reagent from Sigma-Aldrich. SDS-PAGE was carried out under reducing conditions in a 12% gel according to Laemmli et al. [21]. The relative purity, considered as the percentage of the intensity of PspA245 band against the sum of intensity of all other bands in the lane, was determinate by densitometry of SDS-PAGE protein bands in a Biorad GS-800 densitometer and analyzed by Quantity One 4.6.3 software.

3. Results and Discussion

The rfPspA245 was produced using glycerol, a by-product of the Brazilian biofuel industry. It was obtained circa 60 g/L of dry cell weight and 3.0 g/L of rfPspA. Using glycerol as carbon source, the acetate formation was lower than 1.0 g/L during all process (not shown). The biomass production was similar to that previously obtained using glucose as carbon source (not shown).

The Figure 3 shows the rfPspA245 production in high cell density of E. coli using glycerol as carbon source.

Fig. 3. Production of rfPspA245 in fed-batch cultures using glycerol as carbon source. Lane 1: before induction; lanes 2-5: 1-4 h of induction, respectively.

The cell disruption was successfully achieved using a mechanical (high pressure continuous homogenizer) and chemical (detergent Triton X-100) combined method, reaching 96.7% of efficiency.

The first chromatographic sequence, Q-Sepharose followed by IMAC-Sepharose, was used to the purification rfPspA245. The conditions were described above (Figures 1 and 2). The electrophoresis gels of purification are shown in Figure 4 and 5. The results are described in Table 1.

Considering the results described in Figure 4, 5 and Table 1, we can verify that the protein is not present in fraction QF1 but in fraction QF2 is present with higher purity, 44.8%, than in loading fraction 34.9%. In the trade off, the purity was selected instead of recovery and the QF3 fraction was obtained with a purity of 88.1%.

1 2 3 4 5 6 7 8

97 66 45

rfPspA245

Fig. 4. SDS-PAGE of rfPspA245 purification in Q-Sepharose. Lane 1: molecular marker (kDa); lane 2: clarified homogenate; lane 3: Q loading fraction; lane 4: QF1, flow-through; lane 5: QF2, wash 1; lane 6: QF3, elution; lane 7: QF4, wash 2; lane 8: QF5, cleaning.

Fig. 5. SDS-PAGE of rfPspA245 purification in IMAC-Sepharose. Lane 1: IMAC loading fraction; lane 2: NiF1, flow-through; lane 3: NiF2, wash 1; lane 4: NiF3, wash 2; lane 5: NiF4, elution; lane 6: molecular marker (kDa).

Table 1. Purification of rfPspA245 in Q-Sepharose followed by IMAC-Sepharose

Sample Total Protein (mg) Relative Purity rfPspA245 (%)# rfPspA245 (mg) Recovery rfPspA245 (%) Purification Factor

Clarified Homogenate 62010 34.9 21641 100.0 1.0

Q-Sepharose (QF3) 10120 88.1 8916 41.2 2.5

IMAC -Sepharose (NiF4) 5200 96.6 5023 23.0 2.8 (1.1)*

# Calculated by densitometry. * Value between parentheses is the purification factor of this step

In the first chromatographic sequence tested for purification of PspA245, we obtained the necessary purity degree after the IMAC-Sepharose (96.9%). However, the recovery of PspA245 was low (23%). Analyzing the SDS-PAGE (Figure 5) we can observe PspA245 in all fractions of IMAC-Sepharose. So, the chromatographic conditions could be changed in order to increase the recovery.

The second chromatographic sequence tested for purification of PspA245consisted of IMAC-Sepharose followed by Q-Sepharose. The conditions are the same described in Figures 1 and 2. The Figures 6 and 7 and Table 2 show the results. This chromatographic sequence was not indicated to the purification of rfPspA245. The relative purity (79.9%) and the final recovery (9.1%) were lower than using the inverse sequence.

Besides the better results obtained with the first chromatographic sequence (Q-Sepharose followed by IMAC-Sepharose), the use of Q-Sepharose as the first chromatographic step has the advantage of increasing the life-time of the most expensive resin, IMAC-Sepharose.

Fig. 6. SDS-PAGE of rfPspA245 purification in IMAC-Sepharose. Lane 1: molecular marker (kDa); lane 2: clarified homogenate; lane 3: NiF1, flow-through; lane 4: NiF2, wash; lane 5: NiF3, elution.

■ rfPspA245

Fig. 7. SDS-PAGE of rfPspA245 purification in Q-Sepharose. Lane 1: molecular marker (kDa); lane 2: Q loading fraction; lane 3: QF1, flow-through; lane 4: QF2, wash 1; lane 5: QF3, wash 2; lane 6: QF4, elution; lane 7: QF5, cleaning.

Table 2. Purification of rfPspA245 in IMAC-Sepharose followed by Q-Sepharose.

Sample

Total Protein (mg)

Relative Purity rfPspA245 (%)#

rfPspA245 (mg)

Recovery rfPspA245 (%)

Purification Factor

Clarified Homogenate 53872

IMAC-Sepharose (NiF3) 4100

Q- Sepharose (QF4) 2700

44.0 68.5 79.9

23704 2808 2157

100.0 11.8 9.1

1.0 1.5 1.8 (1.2)*

# Calculated by densitometry. * Value between parentheses is the purification factor of this step.

4. Conclusions

The PspA245 was produced in a high cell density cultivation of E. coli, the cells were disrupted with high efficiency and the best sequence for the purification of recombinant PspA was Q-Sepharose followed by IMAC-Sepharose.

The purification processes still need to be improved, especially in the recovery from IMAC chromatography and may be also in the recovery of Q-Sepharose, but it is noteworthy that PspA245 was obtained with purity of 96.6%.

Acknowledgements

This work received the financial support of the Sao Paulo State Research Foundation (FAPESP), under grant 2008/05207-4.

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