GroES homologues from Geobacillus thermopakistaniensis and their applications in protein folding

Accepted Manuscript Title: Cloning and characterization of thermostable GroEL/GroES homologues from Geobacillus thermopakistaniensis and their applications in protein folding Authors: Raza Ashraf, Majida Atta Muhammad, Naeem Rashid, Muhammad Akhtar PII: DOI: Reference:

S0168-1656(17)30267-5 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.05.023 BIOTEC 7905

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

10-4-2017 25-5-2017 31-5-2017

Please cite this article as: Ashraf, Raza, Muhammad, Majida Atta, Rashid, Naeem, Akhtar, Muhammad, Cloning and characterization of thermostable GroEL/GroES homologues from Geobacillus thermopakistaniensis and their applications in protein folding.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2017.05.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cloning and characterization of thermostable GroEL/GroES homologues from

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Geobacillus thermopakistaniensis and their applications in protein folding

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Raza Ashraf1, Majida Atta Muhammad1, Naeem Rashid1,* and Muhammad Akhtar1,2

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1School

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Lahore 54590, Pakistan

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2School

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UK

of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus,

of Biological Sciences, University of Southampton, Southampton SO16 7PX,

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*Corresponding author

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Tel: +92-42-99231534

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Fax: +92-42-99230980

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E-mail: [email protected]; [email protected]

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The authors declare that they have no competing interests.

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Highlights

1) This is the first cloning and characterization of chaperonins from any member of genus Geobacillus.

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2) The chaperonins described in this study were able to refold the denatured insoluble

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aggregates of α-amylase, a biotechnologically important enzyme, from Bacillus

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licheniformis into soluble and active form.

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3) These chaperonins successfully enhanced the thermostability of porcine heart malate dehydrogenase.

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4) Expression of the genes in E. coli cells enhanced the thermotolerance of the host.

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5) Enhancement of soluble production of recombinant alcohol dehydrogenase from

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Bacillus subtilis strain R5 in E. coli, initially produced as insoluble aggregates, was

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achieved by co-expressing the chaperonin gene.

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6) This system can be used for soluble production of recombinant proteins which otherwise are produced in insoluble form in E. coli.

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Abstract

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The chaperonin genes encoding GroELGt (ESU72018) and GroESGt (ESU72017),

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homologues of bacterial GroEL and GroES, from Geobacillus thermopakistaniensis were

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cloned and expressed in Escherichia coli. The purified gene products possessed the

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ATPase activity similar to other bacterial and eukaryal counterparts. Recombinant

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GroELGt and GroESGt were able to refold the denatured insoluble aggregates of α-

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amylase from Bacillus licheniformis into soluble and active form. Furthermore, GroELGt

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and GroESGt successfully enhanced the thermostability of porcine heart malate

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dehydrogenase. Expression of GroELGt gene in E. coli cells enhanced the

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thermotolerance of the host. Furthermore, soluble production of recombinant alcohol

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dehydrogenase from Bacillus subtilis strain R5 in E. coli, initially produced as insoluble

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aggregates, was achieved by co-expressing the gene with GroELGt. Our results implied

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that GroELGt could assist folding of nascent protein in E. coli with the help of host co-

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chaperonin without requiring additional ATP. This system can be used for soluble

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production of recombinant proteins which otherwise are produced in insoluble form in E.

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coli. To the best of our knowledge this is the first report on functional characterization and

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applications of chaperonins from genus Geobacillus.

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Keywords: Chaperonin, co-chaperonin, protein folding, thermotolerance, Geobacillus

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thermopakistaniensis

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Introduction

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Molecular chaperones are a family of multimeric proteins, essential in all the three

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domains of life (Kim et al., 2013), which assist the correct folding and assembly of newly

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synthesized proteins (Bukau et al., 2000), refolding of stress-denatured proteins (Horwich

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et al., 2007), protection of proteins from thermal denaturation (Hartl, 1996; Martin et al.,

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1992), modulation of protein activity (Staniforth et al., 1994) and protein transportation 3

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across cellular membranes (Dierks et al., 1993). A class of chaperones that assists folding

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of proteins, mostly newly synthesized proteins, with the help of ATP is termed as

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chaperonins which is subdivided into two groups. Group I chaperonins are present in

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bacteria, mitochondria and chloroplast (Sigler et al., 1998; Horwich et al., 2007) while

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group II members are found in archaea and eukaryotic cytosol (Lopez et al., 2015). The

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group I chaperonins require a small helper protein, called co-chaperonin, which makes a

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lid like structure on the chaperonin cavity (Bonshtien et al., 2009). The most extensively

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studied member of group I is the bacterial chaperonin named GroEL which is assisted by

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the co-chaperonin named GroES. Group II chaperonins normally do not require a co-

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chaperonin (Shomura et al., 2004; Zhang et al., 2010). Despite the difference in the

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requirement of co-chaperonin, all chaperonins can transit between an open conformation,

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where non-native protein is recognized and encapsulated, and a closed conformation,

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where the substrate protein (non-native protein) is enclosed in cavity and isolated from

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the outer cellular environment. ATP binding and hydrolysis induces this transition (Hartl

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et al., 2011).

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To cope with high demand, industrially important enzymes are being produced in various

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recombinant systems. Escherichia coli is the most popular host for the production of

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recombinant proteins because of ease of transformation and fast growth on inexpensive

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carbon sources (Sezonov et al., 2007; Lee, 1996; Choi et al., 2006; Pope and Kent, 1996).

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However, some of the recombinant proteins produced in E. coli are soluble and active

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whereas others either degrade or make insoluble aggregates called inclusion bodies

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(Yang et al., 2011; Rosano and Eduardo, 2014). Insoluble recombinant protein

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aggregates are mostly inactive. However, their insolubility helps to get relatively pure 4

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proteins which can be refolded to native and enzymatically active conformation (Rudolph

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and Lilie, 1996). For this, the insoluble aggregates need to be solubilized in a denaturant

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and then refolded (Burgess, 2009). Chaperonins can help in refolding the denatured

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protein to native conformation (Motojima and Yoshida, 2015). Furthermore, chaperonins

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are also found to enhance the soluble expression of foreign genes by co-expression with

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the target gene (Martínez-Alonso et al., 2009; Yan et al., 2012; Tian et al., 2016).

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In the present study we report on cloning and expression, in E. coli, of GroEL and GroES

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homologues from Geobacillus thermopakistaniensis whose complete genome sequence

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has been determined (Siddiqui et al., 2014). Applications of these chaperonins for

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production of recombinant alcohol dehydrogenase from Bacillus subtilis R5, previously

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produced in insoluble and inactive aggregates, in active conformation has also been

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described by co-expression with GroElGt gene in E. coli. Furthermore, in vitro refolding of

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insoluble aggregates of α-amylase has been described.

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Materials and Methods

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Chemicals, bacterial strains and growth conditions

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All chemicals were of analytical grade and purchased either from Fluka Chemical

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Corporation, Sigma–Aldrich Company or Thermo Fisher Scientific Inc. DNA polymerase,

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restriction enzymes, cloning vectors, and DNA purification kits were purchased from

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Thermo Fisher Scientific Inc or Novagen-Merck Millipore. Gene specific primers were

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commercially synthesized from Gene Link, Inc. E. coli DH5α strain was used for cloning

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and plasmid preparation, and the E. coli CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA,

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USA) strain was used as a host for the heterologous expression of the gene. G.

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thermopakistaniensis was cultivated in LB (tryptone 1%, yeast extract 0.5%, NaCl 0.5%;

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pH 7.0) medium and grown at 60 °C.

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Construction of recombinant plasmids

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The genes encoding GroELGt and GroESGt were amplified by polymerase chain reaction

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using sequence specific forward (5’-CCATGGCAAAACAAATCAAGTTCAGTGAAG) and

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reverse (5’-TTACATCATGCCGCCCATATCCG) primers for GroELGt, and forward (5’-

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CATATGAAGCCATTAGGCGATCGTATTG)

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TTAGCGGATGACAGCCAAAATATC) primers for GroESGt. Genomic DNA of G.

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thermopakistaniensis was used as template. NcoI site (underlined sequence) was

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introduced in the forward primer of GroELGt and NdeI site (underlined sequence) was

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added in the forward primer of GroESGt. The PCR amplified gene products were cloned

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individually in pTZ57R/T cloning vector. The resulting plasmids were named as pTZ-

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GroELGt and pTZ-GroESGt. The genes were excised from pTZ-GroELGt and pTZ-GroESGt

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plasmids using NcoI + BamHI (for GroELGt) and NdeI + KpnI (for GroESGt) restriction

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enzymes and ligated at the corresponding sites in pETDuet-1 and pRSFDuet-1,

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respectively. The resulting plasmids were named as pETDuet-GroELGt and pRSFDuet-

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GroESGt, respectively.

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Production in E. coli and purification of recombinant GroELGt and GroESGt

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Plasmids pETDuet-GroELGt and pRSFDuet-GroESGt were used to transform E. coli BL21-

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CodonPlus(DE3)-RIL cells. Gene expression for each of the gene was induced by the

and

reverse

(5’-

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addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.1

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mM in individual cultures. After induction, the cells were grown for 5 h at 37 °C and

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harvested by centrifugation at 6,500×g for 10 min. Cells, nearly 1.5 g wet weight from

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each of the 1 L culture, were suspended individually in 50 mL of 50 mM Tris-HCl buffer

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(pH 8.0) and disrupted by sonication. Soluble and insoluble fractions were separated by

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centrifugation at 12,000×g for 30 min. Proteins were analyzed by denaturing

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polyacrylamide gel electrophoresis (SDS-PAGE). Soluble fractions, containing GroELGt

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and GroESGt were partially purified by incubating at 60 °C for 30 min. Further purification

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was carried out by loading partially purified GroELGt or GroESGt to anion exchange column

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(Resource Q, 6 mL). The fractions containing recombinant GroELGt or GroESGt were

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pooled, concentrated by using ultra centrifugal filters (Amicon) and dialyzed against 50

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mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. The dialyzed sample was purified

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by gel filtration column Superdex 200 10/300 GL (GE Healthcare) equilibrated with 50

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mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl.

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Effect of temperature on ATPase activity of GroELGt

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In order to determine the ATPase activity of GroELGt, assays were carried out at various

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temperatures between 40 and 80 °C in 200 μL reaction mixture containing 50 mM Tris-

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HCl (pH 8.0), 2 mM MgCl2, and 150 mM KCl and 10 μg purified GroELGt. After 5 min pre

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incubation of reaction mixture at the respective assay temperature, the reaction was

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started by adding ATP at a final concentration of 1 mM. After incubation for 0, 5, 10, and

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15 min, 20 μL reaction mixture was aliquoted and free orthophosphate was measured by

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malachite green assay kit (BioAssay Systems). The amount of ATP hydrolyzed without

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addition of GroELGt at each temperature was subtracted for the calculation of ATPase

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activity. The amount of free orthophosphate in each reaction was calculated from the

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standard curve.

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Thermostability of recombinant GroELGt

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In order to determine the thermostability of GroELGt, the protein was incubated at 50, 60

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and 70 °C for various intervals of time. The incubations were performed in tightly screw

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capped tubes. The residual ATPase activity was examined at 70 °C. The activity without

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incubation at these temperatures was taken as 100%.

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Structural stability analysis

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In order to measure the structural stabilities, GroELGt and GroESGt were analyzed by

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circular dichroism (CD) spectroscopy using Chirascan-plus CD Spectrometer (Applied

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Photophysics, UK). The CD spectra of the protein solutions were recorded in 10 mM Tris-

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HCl (pH 8.0) in the far UV-range of 190–270 nm. Solvent spectra were subtracted from

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those of the protein solutions.

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In order to investigate the effect of the nucleoside phosphates on the conformation, 5 µM

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of GroELGt was incubated with 0.1 mM ADP or ATP and CD spectra were examined. A

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control experiment without the addition of ATP or ADP was also performed.

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GroELGt and GroESGt assisted refolding of denatured alcohol dehydrogenase

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Yeast alcohol dehydrogenase (6 μM) from Sigma-Aldrich was denatured with 6 M

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guanidine hydrochloride in 1 M Tris-HCl (pH 8.0) and incubated at room temperature for 8

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30 min. The denatured alcohol dehydrogenase was diluted to 0.1 μM with 0.1 M Tris-HCl

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(pH 8.0) containing 1 M KCl, 1 mM MgCl2, 10 µM ZnCl2, 0.4 μM GroESGt and 0.4 μM

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GroELGt, and incubated at 50 °C for 30 min with continuous stirring. The control contained

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all but GroELGt, and GroESGt. The reaction mixture of alcohol dehydrogenase assay

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consisted of 50 mM Tris-HCl (pH 8.0), 50 mM ethanol, 0.2 mM NAD+ and alcohol

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dehydrogenase. The production of NADH was monitored at 340 nm continuously by a

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UV-visible-1601 spectrophotometer with a thermal control unit (Shimadzu, Kyoto, Japan).

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GroELGt and GroESGt assisted refolding of insoluble α-amylase

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In order to properly refold insolubly produced recombinant α-amylase cloned in pET-21a

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(Rashid et al., 2009), the inclusion bodies in insoluble fraction, after lysis of the host cells,

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were resuspended in Tris-HCl (pH 8.0) containing 2% Triton X-100 and sonicated for 1×5

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min. The insoluble fraction, after centrifugation, was resuspended again in the same

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buffer and sonicated in a similar way. This process was repeated four times and the

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inclusion bodies were denatured using 6 M guanidine hydrochloride in 0.1 M Tris-HCl (pH

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8.0) and incubated at room temperature for 30 min. The denatured α-amylase was

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refolded at 50 °C in a similar way as described for alcohol dehydrogenase with overnight

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stirring. The quantitative assay for starch-degrading activity of amylase was based on the

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decrease in absorbance (blue value) of the iodine-amylose complex due to starch

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degradation. The enzyme solution (50 µL) was incubated at 80 °C for 15 min with 90 µL

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of 1% soluble starch solution, and the reaction was terminated by quenching on ice. The

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reaction mixture (10 µL) was taken and mixed with 100 µL of 0.1 N NaOH. This solution

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(100 µL) was mixed with 2 mL of iodine solution (0.005% I2 and 0.05% KI), and the

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absorbance of the mixture was measured at 660 nm (Rashid et al., 2002). The control

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experiment contained all the reagents but no α-amylase.

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Effect of GroELGt and GroESGt on thermal inactivation of commercial malate

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dehydrogenase

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In order to examine protection of commercial malate dehydrogenase against thermal

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inactivation by GroELGt and GroESGt, assay using porcine heart malate dehydrogenase

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(Sigma-Aldrich) was carried out spectrophotometrically at various temperatures (25 to 70

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°C) in the presence or absence of GroELGt and GroESGt in a Shimadzu UV-1601

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spectrophotometer equipped with a thermoelectric cell. The activity was monitored by the

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oxidation of NADH at 340 nm (ɛ 340 nm = 6.22 mM-1 cm-1) and as a result decrease in

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absorbance was monitored. The reaction mixture contained 50 mM Tris-HCl buffer (pH

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8.0), 0.4 mM oxaloacetic acid, 0.2 mM NADH, 2 mM MgCl2, 1 mM ATP, 150 mM KCl,

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0.2 μM malate dehydrogenase, 0.6 μM GroELGt and 0.6 μM GroESGt in a total volume of

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1 mL. A control experiment contained all of the above except GroELGt and GroESGt.

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Before addition of the malate dehydrogenase the reaction mixture was incubated in

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spectrophotometer cell for 3 min in order to acquire the temperature set for the reaction,

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after addition of malate dehydrogenase the mixture was incubated further for 3 min and

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then reaction was started by the addition of NADH.

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In another experiment 0.2 µM malate dehydrogenase was incubated with 0.6 μM GroELGt,

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0.6 μM GroESGt, 1 M KCl, 1 mM MgCl2 and 1 mM ATP at 60 °C. After different time

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intervals samples were taken and residual activity was measured as described above.

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Thermotolerance of host cells harboring pETDuet-GroELGt.

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To determine whether GroELGt enhances the thermal tolerance capacity of the host cells,

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a heat stress was given to E. coli cells carrying pETDuet-GroELGt or pETDuet-1 vector

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(control) and viability of cells was determined. The cells containing either of the above

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two plasmids were grown at 37 °C until an OD660 of 0.4 was reached. The cultures were

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then induced with 0.05 mM IPTG. After 2 h of incubation at 37 °C the cultures were shifted

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to 55 °C and incubated for 1 h. After the heat shock, the cell cultures were diluted (1:50)

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with fresh growth medium. The diluted cultures, 10 μL each, were spread on LB agar

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plates (containing 100 μg/mL ampicillin and 0.05 mM IPTG) and incubated overnight at

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37 °C.

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In another experiment, E. coli cells carrying pETDuet-GroELGt or pETDuet-1 were

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induced with 0.05 mM IPTG when OD660 was 0.4 and incubated at 37 °C for 30 min. The

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cultures were then diluted to an OD660 of 0.1 and shifted at 55 °C in a shaking water bath.

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Cell density was determined by measuring the OD660 at various intervals of time.

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Co-expression of alcohol dehydrogenase and GroELGt genes in E. coli

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In order to carry out the in vivo proper folding of recombinant alcohol dehydrogenase

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(ADHR5) from B. subtilis R5 at the time of production in E. coli, which otherwise produced

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in insoluble and inactive form as shown in Figure 7A (cloned in pET-21a), gene encoding

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ADHR5 was liberated from a previously constructed pTZ-ADHR5 plasmid (Ashraf et al.,

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2017) and cloned in the multicloning site II of pETDuet-GroELGt utilizing NdeI and

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Bpu1102I restriction enzyme sites. The recombinant plasmid was named as ADHR5-

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petDuet-GroELGt and was used to transform E. coli BL21-CodonPlus(DE3)-RIL cells. For 11

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expression experiments, the one of the transformants was grown overnight at 37 °C in

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LB medium containing ampicillin. Dilution (1%) was made in three flasks containing 50

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mL of LB medium. The cells were grown at 37 °C and when OD660 of 0.4 was attained

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two of the flasks were supplemented with 0.07 mM IPTG. One of these two flasks was

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kept at 37 °C while the other transferred to 17 °C. The third flask was transferred to a

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water bath at 45 °C for 30 min and then supplemented with IPTG and shifted to 17 °C

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overnight.

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Results

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Homology comparison

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Amino acid sequence comparison demonstrated that GroELGt and GroESGt were 100%

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identical with molecular chaperones GroEL and GroES from Geobacillus kaustophilus

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(accession # BAD74534 and BAD74533). None of these enzymes has been

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characterized. Among the characterized enzymes, GroELGt and GroESGt displayed

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highest homologies of 68 (accession # WP_011174077) and 62% (accession #

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WP_011174076) respectively, with their counterparts from Thermus thermophilus

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(Taguchi and Yoshida 1993). Multiple alignment of amino acid sequences of GroELGt and

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GroESGt along with their counterparts from T. thermophilus and E. coli demonstrates 13

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non-conserved regions (Regions I‒XIII in supplementary Fig. 1S). Apart from regions III

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and VIII, all the non-conserved regions are found on the outside of the central cavity in

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the enzymes from T. thermophilus contrary to the residues facing the cis-cavity which are

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highly conserved and mostly charged (Shimamura et al., 2004). Conservation of these 12

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residues suggests that their location could be important for the efficient folding of the

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substrate in addition to their role in maintaining the hydrophilicity of the wall. GroEL from

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T. thermophilus is reported to interact with substrate proteins, leading them to adopt their

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correct conformations with the aid of GroES and ATP. The solution structure of the T.

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thermophilus GroEL-GroES complex encapsulating the substrate proteins suggested a

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repulsive interaction between majority of the substrate proteins and the interior wall of the

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cavity, which is suitable for substrate release (Kanno et al., 2009). A similar interaction is

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speculated between GroELGt-GroESGt and substrate proteins.

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Production and purification of recombinant GroELGt and GroESGt

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A high level production of recombinant GroELGt and GroESGt was found in E. coli in the

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soluble form. The recombinant proteins were partially purified by incubating soluble

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fractions at 60 °C for 30 min. Anion exchange and gel filtration chromatographies of the

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soluble fractions after heat treatment resulted in apparently homogeneous proteins when

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analyzed by SDS-PAGE (supplementary Fig. 2S). Approximately 15 mg of GroELGt and

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8 mg of GroESGt were obtained from 1.5 g of E. coli cells (1 L culture).

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ATPase activity of GroELGt

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ATPase activity of GroELGt was assessed colorimetrically by measuring the PO4 released

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from the hydrolysis of ATP using malachite green. Recombinant GroELGt exhibited

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ATPase activity. In order to find out the optimal temperature, the activity was measured

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at temperatures between 40 and 80 °C (Fig. 1A). The activity increased with the increase

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in temperature up to 65 °C (320 nmol/min/mg), which is in good agreement with the

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optimal growth temperature of G. thermopakistaniensis. The ATPase activity of GroELGt

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was higher than thermostable chaperonins of group I from T. thermophilus (100

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nmol/min/mg) and group II from Pyrococcus horikoshii (42 nmol/min/mg).

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Thermal stability assessment assay demonstrated that GroELGt was a thermostable

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protein with a half-life of 100 min at 60 °C and 45 min at 70 °C (Fig. 1B). Incubation at

280

70 °C resulted in total loss of activity after 60 min of incubation.

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Structural analysis by CD spectroscopy

282

The structural stability of GroELGt and GroESGt was analyzed CD spectroscopy at

283

different temperatures ranging from 30 to 80 °C. CD spectra showed that GroELGt

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contains nearly 45% α-helical structures and 15% β-sheets contrary to the GroESGt which

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contains 35% β-sheets and 17% α-helical structures. The shapes of the spectra of

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GroELGt were similar to spectra of homologous proteins from Bacillus stearothermophilus

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and E. coli (Schön and Schumann, 1995). This suggests that secondary structure of the

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GroELGt might be similar to the secondary structures of these proteins with a high degree

289

of α-helical structures.

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The CD spectra of GroESGt at various temperatures showed that there was no significant

291

change in the spectra up to 70 °C indicating that the protein maintains its secondary

292

structures up to 70 °C, higher than the optimum growth temperature of G.

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thermopakistaniensis. However, the CD spetra of GroELGt significantly differed below and

294

above 70 °C. The spectra were similar between 50 to 70 °C indicating a similar secondary

295

structure at 50‒70 °C (Fig. 2). The midpoints of thermal unfolding for GroELGt is 75 °C

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which is comparable to GroEL from E. coli (60 °C) and B. stearothermophilus (70 °C) and

297

lower than GroEL from T. thermophilus (90 °C) (Schön and Schumann, 1995).

298

Effect of ADP or ATP on the secondary structure of GroELGt was analyzed by CD

299

spectroscopy at 30 °C. The results showed that there was no significant change in the

300

CD spectrum in the presence or absence of ADP while a prominent shift in the CD

301

spectrum was observed in the presence of ATP (Fig. 3) indicating a significant change in

302

the secondary structure.

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Refolding of yeast alcohol dehydrogenase

304

Protein refolding activity of GroELGt and GroESGt, in the presence and absence of ATP,

305

was examined at 50 °C using commercial yeast alcohol dehydrogenase as the substrate

306

(Fig. 4). When the denatured alcohol dehydrogenase, with no dehydrogenase activity,

307

was diluted in the refolding buffer in the absence of GroELGt and GroESGt, a few amount

308

of alcohol dehydrogenase was refolded exhibiting a 34% of the original activity. On the

309

other hand presence of GroELGt and GroESGt in the refolding mixture at a molar ratio of

310

3:3:1 (GroELGt:GroESGt: alcohol dehydrogenase) in the absence of ATP resulted in only

311

a 5% regain of the original activity probably due to capturing of the unfolded protein by

312

the added chaperonins. The refolding of captured protein was induced by the addition of

313

ATP to the refolding buffer and approximately 94% of original alcohol dehydrogenase

314

activity was regained (Fig. 4). These results indicated that refolding activity of these

315

chaperonins is ATP dependent.

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Refolding of inclusion bodies of α-amylase from B. licheniformis

317

Recombinant α-amylase from B. licheniformis which was produced in E. coli as insoluble

318

aggregates exhibited a low level of α-amylase activity (Rashid et al. 2009). These

319

inclusion bodies produced in our laboratory were denatured in 6 M guanidine

320

hydrochloride and diluted in the refolding buffer in the absence of GroES Gt. Though the

321

protein did not precipitated after removal of guanidine hydrochloride but no α-amylase

322

activity could be detected after overnight refolding. However, presence of GroELGt and

323

GroESGt in the refolding buffer at a molar ratio of 3:3:1 (GroELGt:GroESGt:α-amylase) in

324

the presence of 1 mM ATP, resulted in a 5-fold increase in α-amylase activity after

325

overnight refolding as compared to the insoluble protein (supplementary Fig. 3S).

326

Protection of porcine heart malate dehydrogenase against thermal inactivation

327

Activity assays in our laboratory showed that porcine heart malate dehydrogenase starts

328

losing activity above 40 °C and a negligible amount of activity was detected at 70 °C.

329

GroELGt and GroESGt when used at 1:3:3 (malate dehydrogenase:GroEL Gt:GroEsGt) ratio,

330

protected the malate dehydrogenase from losing activity at higher temperatures. There

331

was a 100% activity even at 70 °C in the presence of GroELGt and GroESGt at the above

332

mentioned ratio (Fig. 5A).

333

Protection of thermal inactivation of porcine malate dehydrogenase was also examined

334

by incubating the enzyme with GroELGt and GroESGt at 60 °C in the above mentioned

335

ratio for various intervals of time and then measuring the dehydrogenase activity at a fixed

336

temperature of 40 °C. The results showed that malate dehydrogenase rapidly lost its

16

337

activity when incubated at 60 °C whereas presence of GroELGt and GroESGt protected

338

the enzyme from thermal denaturation and there was no decrease in activity even after

339

30 min of incubation at 60 °C (Fig. 5B).

340

Enhancement of thermotolerance of the host cells harboring pETDuet-GroELGt

341

In order to examine if GroELGt protects the host E. coli cells against heat, a thermal stress

342

was given to the cells containing pETDuet-GroELGt or pETDuet-1 (control) and viability of

343

the cells was compared by spreading equal volumes on the selection plates. The cells

344

harboring pETDuet-GroELGt survived at a higher rate compared to the control cells

345

carrying pETDuet-1. E. coli cells carrying pETDuet-GroELGt displayed 4.33×105 viable

346

colony forming units per mL of the culture compared to 9.2×103 for the cells carrying

347

pETDuet-1. Furthermore, the growth of E. coli BL21-CodonPlus(DE3)-RIL cells harboring

348

pETDuet-GroELGt or pETDuet-1 demonstrated that the cells carrying pETDuet-GroELGt

349

were able to grow at 55 °C, though the growth rate was slow compared to the rate at 37

350

°C. In contrast, the cells carrying pETDuet-1 were unable to grow at this temperature (Fig.

351

6).

352

In vivo proper folding of recombinant alcohol dehydrogenase

353

Recombinant ADHR5 has been produced in E. coli in insoluble and inactive form

354

irrespective of the cultivation temperature after gene induction (Fig. 7A). In order to get

355

production of the protein in soluble and active form the corresponding gene was co-

356

expressed with GroELGt. When the incubation temperature after induction was kept 37

357

°C, GroELGt was produced in the soluble form whereas alcohol dehydrogenase was

17

358

produced in the insoluble fraction. We therefore, lowered the incubation temperature,

359

after induction, to 17 °C which resulted in production of more than 60% of the protein in

360

soluble form. Some of the chaperonins require co-chaporonin for their function therefore,

361

we induced the host co-chaperonin by giving a heat shock of 30 min at 45 °C prior to

362

induction and then expression was taken at 17 °C. This resulted in production of all of the

363

recombinant protein in the soluble and active form although overall production of the

364

recombinant protein was lowered (Fig. 7B). These results indicate that GroELGt can be

365

successfully used for in vivo proper folding of recombinant proteins which otherwise fold

366

improperly.

367

Discussion

368

Genome search of G. thermopakistaniensis revealed the presence of bacterial

369

homologues of chaperonins GroEL and GroES. They fall in group I chaperonins which

370

consists of chaperonins and co-chaperonins. The genes encoding chaperonin, GroEL Gt,

371

and co-chaperonin, GroESGt, were cloned and expressed in E. coli. The purified GroELGt

372

exhibited highest ATPase activity at 65 °C, exactly matching the optimal growth

373

temperature of G. thermopakistaniensis (Tayyab et al., 2010), with a half-life of an hour

374

at this temperature. This property can be useful in investigating and optimizing the folding

375

of proteins at higher temperatures.

376

We have studied the effects of these molecular chaperonins on the soluble expression of

377

recombinant alcohol dehydrogenase in E. coli, refolding of denatured alcohol

378

dehydrogenase, refolding of inclusion bodies of α-amylase, protection of malate

379

dehydrogenase from thermal denaturation, and enhancement of thermotolerance of E.

380

coli cells expressing recombinant chaperonin. GroELGt and GroESGt were able to refold 18

381

the denatured commercial yeast alcohol dehydrogenase, inclusion bodies of recombinant

382

α-amylase, and protect thermal denaturation of commercial porcine heart malate

383

dehydrogenase. Furthermore, the expression of GroELGt gene increased the

384

thermotolerance of the host E. coli cells by protecting the denaturation of the host proteins

385

in accordance to the reports in literature (Kumar et al., 2014; Cha et al., 2009; Ezemaduka

386

et al., 2014). Generally, GroEL contributes to stress resistance by binding to a large

387

variety of nonnative proteins. It was shown that about 50% of the E. coli proteins can bind

388

to GroEL (Viitanen et al., 1992). After binding to the small unstructured regions of the

389

substrate proteins, GroEL internalizes and sequesters individual polypeptide chains. The

390

proteins usually gain their native structure after release. The downside of this mechanism

391

is that a substantial amount of GroEL is required to trap a significant fraction of the

392

proteins that unfold under stress conditions. This implies that there is a limit to the

393

protective effect as the level of upregulation of GroEL is limited (Ezemaduka et al., 2014).

394

It can be speculated that production of recombinant GroELGt in high amounts has

395

overcome this limitation and enabled the E. coli cells to grow at a lethal temperature of

396

55 °C. Thus overeproduction of recombinant GroELGt permits the use of E. coli even at

397

unfavorable temperatures. There have been reports on enhancement of thermotolerance

398

of E. coli by overproduction of recombinant GroEL, which were attributed to the refolding

399

activity of GroEL towards misfolded cellular proteins under thermal stress (Seo et al.,

400

2006; Ezemadukaet al., 2014).

401

In vivo folding of recombinant proteins is relatively more complex than the refolding of

402

denatured proteins in vitro. A few reports in literature have demonstrated that co-

403

production of GroEL increased the solubility of recombinant proteins (Nishihara et al.,

19

404

1998; Gupta et al., 2006; Sonoda et al., 2010). When used for in vivo folding of

405

recombinant ADHR5, which otherwise was produced in insoluble and inactive form in E.

406

coli, GroELGt with the help of heat induced co-chaperonin of the host successfully folded

407

the recombinant protein into active form. In combination with the fact that the ADHR5, in

408

the absence of GroEL, was produced in insoluble and inactive form even at low

409

temperature, we speculate that ADHR5 was folded via GroELGt. The nascent ADHR5 chain

410

might have captured by GroELGt via multiple interactions with hydrophobic surfaces on

411

the apical GroELGt domain. After folding in the captured state, the ADHR5 might have

412

released to the cytoplasm with a structure closer to the native state in accordance to the

413

reports in literature (Yan et al., 2012). Enhancement of thermotolerance of the host cells

414

expressing GroELGt gene and in vivo folding of the recombinant protein provide additional

415

support for the protective function of GroELGt.

416

In conclusion, we have shown that GroELGt and GroESGt promote the proper folding of

417

proteins from their chemically denatured states. They also refold and enhance the activity

418

of chemically denatured inclusion bodies. The co-expression of GroELGt and ADHR5

419

resulted in production of soluble ADHR5. Enzymes of prominent properties and importance

420

are sometimes difficult to produce in an active form in a recombinant expression system.

421

The dearth of adequate and effective protein production techniques is one of the key

422

impediments that hinder the biotechnological applications. We have presented here an

423

efficient method for the soluble production of recombinant proteins which would be

424

beneficial for future developments and applications.

425

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Figure legends

550

Fig. 1. Effect of temperature on ATPase activity of GroELGt. A) Comparison of activity

551

at various temperatures (40–80 °C). The reaction mixture was heated at respective

552

temperature for 5 min before adding ATP. B) Thermostability of GroELGt at different

553

temperatures. The enzyme was incubated at respective temperature for various intervals

554

of time and then the residual activity was examined at 40 °C.

555

Fig. 2. Structural stability studies of GroELGt and GroESGt. Far-UV spectra of A) GroELGt

556

and B) GroESGt were analyzed by examining the circular dichroism spectra from 190–270

557

nm.

558

Fig. 3. Circular dichroism studies on GroELGt in the presence and absence of ATP or

559

ADP. Far-UV spectra of GroELGt control (with no addition of ATP or ADP), with the

560

addition of 0.1 mM ADP or ATP.

561

Fig. 4. Relative activities of denatured and refolded alcohol dehydrogenase. Relative

562

activity of: native protein, denatured protein, refolded without the addition of GroELGt and

27

563

GroESGt, refolded with the addition of GroELGt and GroESGt without ATP, and refolded

564

with the addition of GroELGt, GroESGt and ATP.

565

Fig. 5. GroELGt and GroESGt aided thermal protection of porcine malate dehydrogenase.

566

A) Relative enzyme activity of malate dehydrogenase at various temperatures (25–70 °C)

567

in the absence and presence of GroELGt and GroESGt. B) Relative activity of malate

568

dehydrogenase after incubation of various intervals of time at 60 °C in the presence and

569

absence of GroELGt and GroESGt.

570

Fig. 6. Graph showing the enhancement of thermotolerance of host cells expressing

571

GroELGt. Growth of the cells containing empty pETDuet-1 and pETDuet-GroELGt.

572

Fig.7. Coomassie brilliant blue stained SDS-PAGE showing the production of alcohol

573

dehydrogenase (ADHR5) under different conditions. A) Production of ADHR5 in the

574

absence of GroELGt. Lanes: M, standard protein markers;1 and 2, insoluble and soluble

575

fractions of cells grown at 37 °C; 3 and 4, insoluble and soluble fractions of cells grown

576

at 17 °C, lane 5 and 6, insoluble and soluble fractions of cells grown at 17 °C after a heat

577

shock of 30 min at 45 °C. B) Production of ADHR5 in the presence of GroELGt. Lanes: M,

578

standard protein markers; 1 and 2, insoluble and soluble fractions of uninduced cells; 3

579

and 4, insoluble and soluble fractions of induced cells grown at 37 °C; 5 and 6, insoluble

580

and soluble fractions of induced cells grown at 17 °C, lane 7 and 8, insoluble and soluble

581

fractions of cells grown at 17 °C after a heat shock of 30 min at 45 °C.

582

28