Lactobacillus Acidophilus GAPDH Cloning and Expression

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13 Apr 2018

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Cloning, purification, crystallization and preliminary X-ray studies Lactobacillus acidophilus glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Dhaval Patela, Anju Pappachana, Ravikant Palc, Bichitra Biswald and Desh Deepak Singha,b*

The Lactobacillus acidophilus GAPDH has been cloned expressed and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 110.285, b = 112.514, c = 118.361.

Lactobacilli is very well recognized as a health beneficial micro-organism in the human gastrointestinal (GI tract). In this communication, the expression, purification, crystallization and primary X-ray characterization of GAPDH from Lactobacillus acidophilus is reported. The protein was crystallized using micro batch under oil method in 60-well terasaki plates using 10% w/v Polyethylene glycol 1000, 10% w/v Polyethylene glycol 8000 as precipitant for crystal formation. X-ray diffraction data was collected to 2.52 Å resolution and further processed in the orthorhombic space group P 21 21 21. There were four protein subunits found in the asymmetric unit, which gave a Matthews coefficient VM of 2.51 , corresponding to 51.1% solvent content.s

Lactobacillus; GAPDH; expression; purification; crystallization.

  1. Introduction

Lactobacilli belong to lactic acid bacteria (LAB) which are a diverse group of gram-positive, anaerobic/microaerophilic, non-sporulating, low G+C content bacteria belonging to the phylum Firmicutes (Pot et al. ,1994). L. acidophilus NCFM, marketed as probiotic microbe is widely used in fermented dairy products and dietary supplements (Sanders & Klaenhammer, 2001). Adhesion to host tissues is a prerequisite first step of bacterial colonization and is generally mediated by cell surface adhesion proteins. Binding of microbial adhesins to epithelial cells receptor plays a major role for the adherence of lactobacilli to epithelial cells of mucosal surfaces (Kinoshita et al., 2007).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) an essential intracellular glycolytic enzyme that catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate along with the presence of inorganic phosphate and cofactor NADH or NADPH (Harris & Waters, 1976). GAPDH is also a principal "moonlighting" protein/multifunctional enzyme with additional roles like activation of transcription (Zheng et al., 2003), initiation of apoptosis (Hara et al., 2005), and pathogen–host interactions (Bergmann et al., 2004). GAPDH is reported as secreted or surface associated protein in L.acidophilus (Johnson et al., 2013) though it is a housekeeping gene that does not possess any signal sequences, cell wall-anchoring motifs, or hydrophobic membrane-spanning regions that could target it to a secretion pathway. The mechanism that contributes it to surface location is still unknown. GAPDH on Streptococcal surface (SDH) from group A streptococcus exhibits multiple binding activities to plasminogen (Winram and Lottenberg 1996; D’Costa and Boyle 2000), fibronectin, lysozyme, myosin and actin (Pancholi and Fischetti 1992). Surface-localized GAPDHs has also been showed to bind transferrin of Staphylococcus aureus and Staphylococcus epidermidis (Modun and Williams 1999); whereas Candida albicans GAPDH binds to fibronectin and laminin (Gozalbo et al. 1998). It is also very well known that GAPDH mediates microbe–host interactions in lactic acid bacteria (Kinoshita et al., 2008a)

  1. Materials and methods
    1. Molecular cloning, expression and purification of recombinant GAPDH

Lactobacillus acidophilus strain was procured from National Dairy Research Institute (NDRI), Karnal. Genomic DNA was isolated using standard procedure (Ulrich et al., 2001) and strain was confirmed through 16s RNA sequencing. The GAPDH gene (LBA0698) was amplified by polymerase chain reaction (PCR) using primers GapdhFwd (5’-GTAAGATCTGATGACAGTTAAAATTGGT-3’), which contains a Nde I restriction site (bold) followed by the first 18 nucleotides of the open reading frame (ORF) and GapdhRev (5’-GTAGGATCCTTAAAGAGTAGCAAAGTG -3’) which contains a Bam HI restriction site (bold), stop codon (underlined) followed by the reverse complement of the last 15 nucleotides of the ORF. The ORF was sub cloned in pET28a (Novagen, San Deigo, USA) for expression as a recombinant GAPDH with N-terminal 6X-HIS tag. Both the PCR product and the plasmid pET28a were digested with NdeI and BamHI restriction enzymes (Fermantas INC, USA). The ligation mixture was transformed into chemically competent Escherichia coli DH5a and insertion was confirmed by PCR using above primers. Using the Qiagen Miniprep kit, the plasmid containing the insert was isolated. The identity of the insert was confirmed by sequencing. The final construct (pET28a-gapdh) encodes the full-length GAPDH protein with a N-terminal His tag MGSSHHHHHH. pET28a-gapdh was transformed into chemically competent E. coli BL21λ DE3 Rosetta cells for expression. Cultures of E. coli BL21λ DE3 Rosetta cells carrying pET28a-gapdh were grown overnight in 20 ml LB medium supplemented with 50 mg/lt kanamycin, 34 mg/lt chloramphenicol and were used to inoculate secondary culture of 500 ml LB medium also supplemented with 50 mg/lt kanamycin and 34 mg/lt chloramphenicol. When O.D. of secondary culture reached 0.6, the protein expression was induced using 0.2 mM IPTG. After overnight growth at 20C with shaking at 200 RPM, the cells were pelleted by centrifugation. The cells were then re-suspended in 50 mM TRIS pH 7.5, 100 mM NaCl, 1mM PMSF, 0.1% triton-X and lysed by sonication using cycle 5sec on and 10sec off for 2min (Vibra-cell sonics, CT, USA). Soluble fraction was obtained by centrifugation and the crude lysate was loaded onto a Ni–NTA resin column (Sigma-Aldrich, India) previously equilibrated with 50 mM TRIS pH 7.5, 100 mM NaCl, 20 mM imidazole. The column was washed with 50 mM TRIS pH 7.5, 100 mM NaCl, 30 mM imidazole and bound protein was eluted with 50 mM TRIS pH 7.5, 100 mM NaCl, 250 mM imidazole.

  1. Crystallization screening and optimization.

The purified gapdh was concentrated using Amicon ultrafiltration unit using 10 kDa molecular-weight cut-off membrane (Millipore, Massachusetts, USA). The final protein was dialysed against 50 mM TRIS pH 7.4 using 14000 Da molecular weight cut-off dialysis tubing cellulose membrane (Sigma-Aldrich, India). Crystallization trials were carried out with 2-10 mg/ml protein, at different pH range (7.0-8.0). Crystallization experiments were carried out in a 60-well plate using under oil micro batch technique (Grenier, Germany) using reservoirs filled with each of screening condition. Trial conditions were prepared corresponding to the commercially available crystallization screens Crystal Screen, Crystal Screen 2 and Index Screen (Hampton Research, USA). For each well, droplets with a 1:1 protein:precipitant ratio layered with 1:1 mixture of silicon and paraffin oil was setup and plates were incubated at 297K.

  1. Crystallization screening and optimization.

Crystals were mounted in nylon loops (Hampton Research), dragged briefly through parafï¬Ân oil. No separate cryoprotectant was used. Data were collected at 100 K using a Cu-Kα X-ray beam generated by X-ray generator, a Rigaku FR-E+ (Fig. 1) and was processed using imosflm (Powell HR et al., 2013).

  1. Results and discussion

Full-length gapdh protein with a N-terminal 6X-His tag was expressed from the plasmid pET28a-gapdh and purified using metal affinity chromatography. The purity of sample was assessed using 15% commasive stained SDS-PAGE (Fig. 2). The identity of protein was confirmed through MALDI analysis.

Crystal Screen, Crystal Screen 2 and Index Screen produced many trial solution containing micro crystals of different size and shape which grew within a week or two after crystallization setup. Two particularly promising conditions that gave diamond-shaped crystals were conditions 10% w/v Polyethylene glycol 1,000, 10% w/v Polyethylene glycol 8,000 and 25% w/v Polyethylene glycol 1,500 of the crystal screen 2 and index screen respectively. These conditions were further optimized. Diffraction quality crystals were obtained by micro batch under oil method using 10% w/v Polyethylene glycol 1,000, 10% w/v Polyethylene glycol 8,000 as precipitant were used for X-ray data collection at cryotemperature.

The orthorhombic crystals belonged to space group P212121 and diffracted to 2.51 Å resolution at 103 K (one of the oscillation images is shown in Fig. 3); data-processing statistics are mentioned in Table 1. The unit cell parameters were a = ,b= ,c= Å. A total of 1063508 reflections were collected, of which 50257 were unique; the completeness of the diffraction data was 99.1 %. Assuming the presence of four monomeric molecules (with a calculated molecular weight of 36,495 Da each) per crystal asymmetric unit, the calculated Matthews coefficient and solvent content are 2.51 Å3 Da-1 and 51.1%, respectively (Matthews, 1968).

GAPDH have been reported earlier as molecule involved in adhesion performing non-glycolytic moonlighting function (Turpin et al.,2012; Kinoshita et al., 2008a; Kinoshita et al., 2008b; Hurmalainen et al., 2007; Ramiah et al., 2008; Sanchez et al., 2009; Glenting et al., 2013). Previous report on LBA0698 (GAPDH) indicates that its a secreted/ surface associated protein (Johnson et al., 2013). This is the first attempt towards its structural studies which we believe will throw light on its moonlighting function. Further work on structure determination of the protein is in progress.

  1. Diffraction image of GAPDH.

  1. SDS–PAGE profile of the purified gapdh. The gel was stained with Coomassie Brilliant Blue R-250. Lane 1, molecular-mass marker (labelled in kDa); lane 2, purified GAPDH.

  1. Crystal of L.acidophilus GAPDH grown using 10% w/v Polyethylene glycol 1,000, 10% w/v Polyethylene glycol 8,000 as precipitant.

  1. Data collection statistics for GAPDH

Values for the outer shell are given in parentheses.

 

Diffraction source

Cu Kα

Wavelength (Å)

1.54178

Temperature (K)

100

Detector

R-AXIS IV++

Crystal-detector distance (mm)

210

Rotation range per image (°)

0.5

Total rotation range (°)

180

Space group

P212121

Unit-cell parameters a, b, c (Å)

a=110.285, b=112.514, c=118.361

α, β, γ (°)

α = β = γ FORMTEXT = 90

Resolution range (Å)

50-2.52 (2.61-2.52)

Total No. of reflections

1063508

No. of unique reflections

50257

Completeness (%)

99.9 (99.8)

Redundancy

1.1

{I/σ(I)}

10.296 (4.36)

Rmerge

0.16 (0.45)

Matthews coefficient ( Å3 Da-1 )

2.51

No. of molecules in asymmetric unit

4

Overall B factor from Wilson plot (Å2)

 
  • - The authors thank the Distributed Information Sub-Center (DISC) at Indian Institute of Advanced Research funded by Department of Biotechnology (DBT), Government of India for financial support and assistance to carry out the work. Authors also wish to thank The Puri Foundation for Education in India for providing support and opportunity to carry out the work. The X-ray diffraction facility at National Institute of Immunology (NII), New Delhi was used for data collection which was established with financial support from the DBT.

References

Bergmann, S., Rohde, M. & Hammerschmidt, S. (2004). Infect Immun 72, 2416-2419.

D'Costa, S. S. & Boyle, M. D. (2000). Methods 21, 165-177.

Glenting, J., Beck, H. C., Vrang, A., Riemann, H., Ravn, P., Hansen, A. M., Antonsson, M., Ahrne, S., Israelsen, H. & Madsen, S. (2013). Microbiol Res 168, 245-253.

Gozalbo, D., Gil-Navarro, I., Azorin, I., Renau-Piqueras, J., Martinez, J. P. & Gil, M. L. (1998). Infect Immun 66, 2052-2059.

Hara, M. R., Agrawal, N., Kim, S. F., Cascio, M. B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J. H., Tankou, S. K., Hester, L. D., Ferris, C. D., Hayward, S. D., Snyder, S. H. & Sawa, A. (2005). Nat Cell Biol 7, 665-674.

Harris, J. I. &Waters, M. (1976). The Enzymes, edited by P. D. Boyer, pp. 1–49. New York: Academic Press.

Hurmalainen, V., Edelman, S., Antikainen, J., Baumann, M., Lahteenmaki, K. & Korhonen, T. K. (2007). Microbiology 153, 1112-1122.

Johnson, B., Selle, K., O'Flaherty, S., Goh, Y. J. & Klaenhammer, T. (2013). Microbiology 159, 2269- 2282.

Kinoshita, H., Uchida, H., Kawai, Y., Kitazawa, H., Miura, K., Shiiba, K., Horii, A. & Saito, T. (2007). J Appl Microbiol 102, 116-123.

Kinoshita, H., Uchida, H., Kawai, Y., Kawasaki, T., Wakahara, N., Matsuo, H., Watanabe, M., Kitazawa, H., Ohnuma, S., Miura, K., Horii, A. & Saito, T. (2008a). J Appl Microbiol 104, 1667-1674.

Kinoshita, H., Wakahara, N., Watanabe, M., Kawasaki, T., Matsuo, H., Kawai, Y., Kitazawa, H., Ohnuma, S., Miura, K., Horii, A. & Saito, T. (2008b). Res Microbiol 159, 685-691.

Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.

Modun, B. & Williams, P. (1999). Infect Immun 67, 1086-1092.

Pancholi, V. & Fischetti, V. A. (1992). J Exp Med 176, 415-426.

Pot, B., Ludwig, W., Kersters, K. and Schleifer, K.H. (1994) In Bacteriocins of Lactic Acid Bacteria eds De Vuyst, L. and Vandamme, E.J. pp. 55±57. Blackie Academic and Professional.

Powell, H. R., Johnson, O. & Leslie, A. G. (2013). Acta Crystallogr D Biol Crystallogr 69, 1195-1203.

Ramiah, K., van Reenen, C. A. & Dicks, L. M. (2008). Res Microbiol 159, 470-475.

Sanchez, B., Schmitter, J. M. & Urdaci, M. C. (2009). J Mol Microbiol Biotechnol 17, 158-162.

Sanders, M. E. & Klaenhammer, T. R. (2001). J Dairy Sci 84, 319-331.

Turpin, W., Humblot, C., Noordine, M. L., Thomas, M. & Guyot, J. P. (2012). PLoS One 7, e38034.

Winram, S. B. & Lottenberg, R. (1996). Microbiology 142 ( Pt 8), 2311-2320.

Ulrich, R. L. & Hughes, T. A. (2001). Lett Appl Microbiol 32, 52-56.

Zheng, L., Roeder, R. G. & Luo, Y. (2003). Cell, 114, 255–266.



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