Mol Biotechnol DOI 10.1007/s12033-014-9783-8

RESEARCH

Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1 Sanjay Kumar

Ó Springer Science+Business Media New York 2014

Abstract Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with [73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)2SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5–5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa. Keywords HIC-1  GC-rich  Cloning  Protein expression  Tumor suppressor gene

S. Kumar (&) Biomolecular Science Centre, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Bld 20, 4110 Libra Drive, Orlando, FL 32816, USA e-mail: [email protected]

Introduction Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor gene located on chromosome 17p13.3 and telomeric to p53, which transcriptionally activates its expression by binding to its promoter [1]. HIC-1 hypermethylation leads to an increased cell proliferation and tumorigenesis [2, 3]. Previously, we showed that P53 inactivation may be an early event in glial tumorigenesis whereas loss of HIC-1 involved in progression from lower grade to higher grade of malignancy in glial tumors [4]. HIC-1 known to cause differentiation and its abrogation might lead to the de-differentiation seen in higher grades of astrocytic tumors [5]. Genomic structure of HIC-1 revealed three exons (exon1a, exon-1b, and exon-2) gives rise to several alternatively spliced variants with common 30 -terminal exon-2 and different 50 -terminal exons (exon-1a, exon-1b or exon-1c) in different human and mouse tissues [6]. Exon-1a is noncoding and is associated with the major GC-rich promoter whereas exon 1b is a coding exon with TATA promoter [7, 8] (Fig. 1). HIC-1 encodes for 714 residues protein with five Kruppel-like C2H2 zinc finger motifs and N-terminal broad complex, tramtrack, and bric-a-brac/pox virus and zinc finger (BTB/POZ) domain found in actin-binding proteins or transcriptional regulators involved in chromatin modeling [9]. Transcriptional start site was identified within 40-bp downstream of a TATA box sequence, preceding an untranslated first exon. The putative ATG and the Zin (Zinc finger N-terminus) domain are located in second exon containing a translational stop site TAG upstream to polyadenylation site [10]. At present little is known about structural and functional aspects of HIC-1, its various domains and moreover it’s binding specification to different promoters or protein

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A

TAG poly A tail

ATG

P2(G-C)

P1(G-C)

P0(TATA)

1a

1c

gagag BTB/ POZ

1b

ZF

2

Exon-2

B

R2

F2

1833bp

BTB/ POZ

BamHI

ZF

SacI

XhoII 372bp F1

445bp 817bp R1

Fig. 1 Schematic structure of HIC-1 gene locus with different promoters and exons codes for different transcripts. a P0, P1, and P2 are promoters and exon 2 is common for all the transcripts (black box). gagag, splicing acceptor site for all the spliced transcripts. Large boxes represent the coding regions from different transcripts coding

for the Bric-a-brac, Tramtrack, Broad complex/Pox virus and Zinc finger (BTB/POZ), and zinc finger (ZF) domains of HIC-1 protein. b Full coding length exon 2 of *2.2 kb with location of F1, R1, F2, and R2 primers and restriction endonuclease BamH1, XhoII, and Sac1 sites

complexes for transcriptional activity are grossly unknown. HIC-1 with high GC content ([73 %) poses daunting task to amplify, sequence, clone and express full-length protein for above studies so requirement was felt. We described here the strategy for cloning and expression of high GCrich full-length HIC-1 in pGEM-T easy vector (cloning vector) followed by subcloning into pPROEX-HTb (expression vector) and later its expression in Escherichia coli.

site as docking site of restriction enzymes for efficient cleavage. Sequence of F1 (forward) and R1 (reverse) were 50 CGTA GGA TCC GAG TGT GCT GGG CAG ACG 30 and 50 CGTA GAG CTC GGC GAG AGG CGG CTC CTT GTA G 30 ,respectively. F2: 50 CGTA GGA TCC TAC CTG CAG ATC CCC GAC CTC GT 30 and R2: 50 CGTA GAG CTC CAG CAG CGA CAG AGC GG 30 . The underlined sequences represent restriction sites of BamHI in forward primers and SacI site in reverse primers. cDNA Synthesis

Materials and Methods Primer Design The primers were designed using Primer-Select from DNASTAR and Primer3 software (http://frodo.wi.mit.edu/ cgi-bin/primer3/primer3_www.cgi). The average length of primers were 22–25 bases and care was taken to avoid primer complementarity especially at the 30 ends which often results in the formation of primer-dimers and hairpin structures with high GC content [11]. Four bases at the 30 end of the primers with more than 50 % C/G and extreme last base was either C or G. Adapter of 10 bases were added to 50 -end of primers designed to clone in pProEXHTb using restriction sites for BamHI (GGATCC; forward primers) and SacI (GAGCTC; reverse primers) as per multiple cloning site (MCS). The adapter attached was in reading frame of HIC-1 which coincides with translation start site of vector. An extra 4 base overhang (combination of A, T, G, and C) was added to the 50 -end of the restriction

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RNA was isolated from PBMCs using RNAqueous-4PCR Kit (Ambion Inc., Austin, Texas). In brief, cell lysis buffer was added to the PBMC pellet (500 ll/106 cells) and vortex vigorously until lysate became homogeneous. Equal volume of 64 % ethanol solution was added and the mixture was transferred to filter cartridge assembled in collection tube and centrifuged at 12,000 rpm until the mixture passed through the filter. After washing the column, RNA was eluted in a fresh collection tube using pre-heated (95–100 °C) RNase free water. First strand cDNA was synthesized by random decamers using RETROscript Kit (Ambion Inc., Austin, Texas) as per manufacturer’s protocol. The RT-mixture was stored at -20 °C. A negative control without reverse transcriptase was also performed. PCR Conditions PCR performed with primer-pairs against HIC-1 and GAPDH (forward: 50 CCA AGG TCA TCC ATG ACA

Mol Biotechnol

ACT TTG GT 30 and reverse: 50 TGT TGA AGT CAG AGG AGA CCA CCT G 30 to look for any DNA contamination). These intron-spanning GAPDH primers were from base 4,703 to 4,728 (forward) and 5,252 to 5,276 (reverse) (Accession No: AY340484) and amplify a product of 381 bp from cDNA and 574 bp from genomic DNA. A combination of strategies using co-solvent (glycerol), denaturing agents (formamide, dimethyl-sulfoxide (DMSO), ammonium sulfate, magnesium) with different heat stable polymerases had been tried out during standardization. Recombinant Pfu DNA polymerase (MBI Fermantas, Hanover, MD) was used for all RT-PCR reactions. The PCR reaction was standardized with 1.5 U polymerase in NH4SO4 containing PCR reaction buffer (MBI-Fermentas) with 10 % DMSO and 1.5 mM Mgcl2. PCR reaction conditions were as follows: Initial Denaturation at 94 °C for 3 min, Denaturation 94 °C, 45 s, Annealing 60 °C for 1 min and Extension 72 °C, 2 min for first two cycles followed by Denaturation 94 °C 45 s, Annealing 67 °C 1 min, Extension 72 °C 2 min for 35 cycles. An initial step of heat denaturation (94 °C for 3 min) was given in every PCR reactions. As the primers had 10 base adapter sequence at the 50 end, the initial two cycles were performed at a lower annealing temperature (*Tm of gene specific part of the primers) followed by 35 cycles at higher annealing temperature (*Tm of whole adapter primers). Construction of Recombinant Cloning Plasmid Products were resolved on agarose gel and eluted using QIAquick Gel Extraction Kit (Qiagen). PCR were performed with proofreading Pfu DNA polymerase gave blunt end PCR products so treatment was done before going for T–A cloning. Briefly, 1–7 ll of the gel-purified PCR fragment, 1 ll of Taq DNA polymerase 10 9 Reaction Buffer with MgCl2 and 0.2 ll of dATP (10 mM) were taken into a microfuge tube. 1.6 ll (*5U) of Taq DNA polymerase (MBI Fermantas, Hanover, MD) was added and the final volume was made to 10 ll with MQ water. The components were properly mixed, briefly spun down, and incubated at 70 °C for 15–30 min. 1–3 ll of this dAtailed DNA fragment was used in the ligation reaction. The dA-tailed DNA fragment was cloned in pGEM-T Easy Vector (T–A cloning vector for sequencing and subcloning) as per manufacturer’s protocol. Briefly, The dA-tailed DNA product was taken as 3:1 molar ratio (PCR product:T-vector) in the ligation reaction. The ligation reaction mix was incubated at 4 °C in a temperature-controlled water bath overnight. 5 ll of this ligation mixture was transformed into E. coli strains DH5a and plated overnight. Next day the antibiotic resistant white colonies (in LacZ selection plate) were picked, sub-cultured in 2xTY media

(containing antibiotic). The positive clones were screened by colony PCR and/or by plasmid isolation and restriction digestion. Sequencing The clones with proper size insert were sequenced using base deoxyinosine 50 -triphosphate [dITP] or 0.2 mM 7-deaza-deoxyguanosine 50 -triphosphate [dGTP] from Boehringer Mannheim, Indianapiolis, IN, 1 M betaine (sigma) with 10 % DMSO by automated DNA sequencer (ABI PRISMÒ) using the universal sequencing primers M13. The sequences were then checked by NCBI BLAST and PSI-BLAST. Homology searching was also performed with the HIC-1 mRNA sequence by DNASTAR BLAST 2 SEQUENCES (http://www.ncbi.nlm.nih.gov/blast/bl2seq/ wblast2.cgi). Construction of Recombinant Expression Plasmid F1RI (short) plasmids isolated from the positive T–A clones and double digested with the enzymes BamHI and XhoII whereas F2R2 (long) with XhoII and SacI to clone in pProEx-HTb. Simultaneously the expression vector also double digested with BamHI and SacI. Digested products were resolved on 2 % agarose gel and eluted using QIAquick Gel Extraction Kit (Qiagen). Column purified digested inserts and vector was then subjected for the ligation. The ligation reaction mix was incubated at 4 °C in a temperature-controlled water bath overnight. 5 ll of this ligation mixture transformed into E. coli strains DH5a, BL21 and BL-21 Codon Plus-RP and plated on antibiotic containing TY agar plates and incubated overnight at 37 °C. Next day the antibiotic resistant white colonies were picked, sub-cultured in 2xTY media (containing antibiotic). The positive clones were screened by colony PCR and/or by plasmid isolation and restriction digestion. Expression vector pHAT-10 (Qiagen) was tried first for expression which showed no induction in different E. coli strains (DH5a, BL-21, and BL-21 Codon Plus-RP). A strong trc-promoter containing expression vector pProEXHTb was then used to clone and express the fragments. Expression of Cloned Fragment in E. coli Positive clones were cultured overnight in 5 ml 2xTY medium containing antibiotic at 37 °C. Glucose (1 %) was added to the culture medium to suppress protein expression of clones carried Lac promoter. A subculture was made in 25 ml 2xTY media from overnight culture and grown at different temperatures (25, 30, and 37 °C) with shaking till OD600 reached 0.8–1.0. Glucose and antibiotics were added as required. Cells were harvested at 1,000 g for 10 min at

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4 °C and resuspended with fresh 2xTY media. IPTG (1 mM) was added to the media for induction. 1.5 ml of the culture media was collected in microfuge tube at the time points 2, 4, 6, 8, 12, and 24 h, and kept at 4 °C. The culture samples were spun down at 5,000 g for 10 min at 4 °C. The supernatant was then stored at -20 °C. 200 ll PBS (pH 7.4) with 1 mM EDTA was added to the cell pellet, resuspended, and kept on ice for 10 min. The cells were spun down as before and the supernatant (for periplasmic fraction) was stored at -20 °C. 200 ll PBS (pH 7.4) was added to the cell pellet, resuspended, and kept in boiling water for 5–10 min. The cells were spun down at 10,000 g for 10 min at 4 °C and the supernatant (for cytoplasmic fraction) was stored at -20 °C. All the fractions at different time points were then loaded on SDS-PAGE and analyzed for the level of protein expression. Finally proteins were imunoblotted against HIC-1 antibody from abcam using 1:4,000 dilution.

vector pProEX-HTb (Fig. 3a) also double digested with the same enzymes (BamHI, SacI) and the backbone was gel purified. Digested fragments were ligated and transformed into BL21-Codon Plus-RP strain of E. coli. Positive clones were picked, inoculated, prepared the plasmids, and digested with BamHI and SacI to confirm the clone (Fig. 3b). Cloning of Long Fragment F2R2

Results

Amplification of large fragment using pfu polymerase gave 1,853 bp fragment (Fig. 4a) and directly cloned into pPROEX-HTb. Double digestion of F2R2 fragment and pPROEX-HTb with BamHI and SacI gave bands of 1,843 bp and 4.7 kb ,respectively, (Fig. 4b) as F2 primer tagged with BamHI whereas R2 with SacI in their adapter sequence. The digested fragments were ligated and later transformed into E. coli BL21-Codon Plus-RP. Positive clones were verified by digesting with BamHI and SacI resulted in 1,843 bp band (Fig. 4c) which was later confirmed by sequencing.

Schematic Structure of HIC-1 and Cloning Strategy

Full-Length HIC-1 Clone

Exon-2 is common in all transcripts and only coding sequence of HIC-1 with start and stop codon. Earlier efforts of full-length cloning had failed due to high GC content and its length. We attempted to clone in two fragments, F1R1 (short) and F2R2 (long) later ligating them to achieve full-length clone (Fig. 1b).

Digesting cloned plasmid F1R1and F2R2 with BamHI, SacI gave 827 and 1,843 bp bands ,respectively, which were eluted and ran on gel (Fig. 5a). The short and long inserts were further digested with XhoII (single site in exon-2) yielded two fragments of size 377 and 450 bp from F1R1 and 1,825 bp from F2R2 as restriction site of XhoII was 7 bp downstream to BamHI restriction site of long fragment (Fig. 5b). The 377 bp has sticky end for BamH1 toward 50 end and XhoII on 30 end whereas F2R2 yields sticky end for XhoII toward 50 end and SacI on 30 end. Triple ligation was done with 377 bp fragment obtained with BamHI and XhoII, 1,825 bp from XhoII and SacI and back bone of pPROEX-HTb digested with BamHI and SacI. The ligated product was transformed into BL21Codon Plus-RP and later isolated the plasmid from ampicillin resistant colonies the next day. They were later digested with BamHI and SacI which gave 2.2 kb band (Fig. 5c). Schematic representation of full-length cloning of HIC-1into pPROEX-HTb (Fig. 5d).

Cloning of Short Fragment F1R1 cDNA were isolated from PBMCs and PCR done using F1 (forward), R1 (reverse) primer for short fragment. Amplification with pfu polymerase gave 837 bp fragment (Fig. 2a). Tailing procedure was done to attach dA overhangs as discussed in the methodology and later ligated with pGEM-T easy vector containing dT overhang (Fig. 2b). The ligated mixture transformed into E.coli DH5a and plated on ampicillin containing agar plate with X-gal and IPTG as pGEM-T Easy vector has both lacZ and b-lactamase genes. The positive white colonies were picked individually next day, grown, and isolated the plasmids. EcoRI digestion of the cloned plasmid resulted in 861 bp insert (Fig. 2c) which was larger than the PCRamplified products by 24 bp, since EcoRI restriction sites in plasmid vector were 9 and 15 bp flanking T–A cloning site. Screening and sequencing confirmed specificity of clone. The T–A clones double digested with BamHI and SacI gave 827 bp insert and was smaller than original PCR product as restriction sites were present into the adapter sequence of forward and reverse primer. The expression

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Expression of Full-Length HIC-1 Protein in BL21-Codon Plus-RP An attempt was made to express full-length clone of HIC-1 in DH5a and BL21-Codon Plus-RP strain of E. coli induced with different concentrations of IPTG (0.5–5.0 mM) for time points 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone expressed in BL21-Codon Plus-RP at 1 mM concentration

Mol Biotechnol

B

A M

1

1 kb 837 bp 500 bp

C

M

D

UD

3 kb

1 kb

861 bp

500 bp

Fig. 2 Cloning of F1R1 (short) fragment in T–A cloning vector. a PCR with F1 (forward) and R1 (reverse) primer amplification gave band of 837 bp. b Schematic representation of cloning vector PGEM-

T Easy vector. c Digestion of PGEM-T plasmid with EcoRI dropped insert of 861 bp. M DNA marker, Lane 1 PCR product, D digested plasmid, UD undigested plasmid

of IPTG for 8 h at 37 °C gave prominent band of 74 kDa (Fig. 6). Uninduced strain also shows some level of expression due to the Tryptophan and Lac (trc) promoter of pPROEX-HTb known to be a leaky promoter. The untransformed strain did not showed any expression of protein.

and 30 end passing further deep after crossing the F2 site. The idea was to digest both long and short fragment with respective restriction enzymes and triple ligating the desired fragments with pProEX-HTb vector and transformed into E. coli. The amplification of targets rich in GC content or ones that can form secondary structure often require laborious work to optimize the amplification assay. Moreover, amplification may result in products derived from region other than target DNA as a result optimisation of magnesium concentration, buffer pH, denaturation and annealing times and temperatures, cycle number is useful. A variety of additives and enhancing agents can be included in PCR amplifications to increase yield, specificity, and consistency. Agents include dimethyl sulfoxide (DMSO), N,N,Ntrimethylglycine (betaine), formamide, glycerol, nonionic detergents, bovine serum albumin, polyethylene glycol, and tetramethylammonium chloride [12–14]. HIC-1 has [73 % GC content which made its amplification difficult due to more stable secondary structures in template and

Discussion All important domains and predicted immunogenic regions (if intended to raise antibody) are present in exon-2 which is common in all transcripts. The objective was to amplify full-length high GC-rich exon-2 codes for 714 amino acids including the start and stop codon. An attempt to clone and express full-length HIC-1 with amplified product from F1R2 primer pair was never achieved due to its large size and high GC content which confers very stable secondary structure. We ended up having F2R2 as our longest clone and another small clone F1R1 containing 50 end of exon 2

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Cloning Sites

A

B

M

D

UD

3 kb

1 kb

827 bp

500 bp

Fig. 3 Cloning of short fragment in expression vector. a Schematic representation of expression vector pPROEXHTb. b Digestion of pPROEXHTb with restriction endonuclease BamHI and SacI dropped

insert of 827 bp. M DNA marker, D digested plasmid, UD undigested plasmid

greater propensity of mispriming using primers with high GC content. Primers of high melting temperature (62–72 °C) were chosen to allow relatively high annealing temperatures and enhance specificity. Initially without cosolvents, nonspecific products were amplified and standardization done by the addition of 2–10 % glycerol, 1–5 % formamide, and 2–10 % DMSO. Glycerol improved thermal stability of DNA polymerase and amplification of high GC-templates, whereas formamide impart specificity of PCR at lower denaturation temperatures [14]. Although, 10 % DMSO decreases the DNA polymerase activity by 50 % but can also reduce secondary structures and presumed to lower the Tm which can be useful for GC-rich templates [12]. Glycerol and/or formamide were not

specifically amplifying HIC-1. Further standardization using 1.5 U DNA polymerase with (NH4)2SO4 containing buffer gave specific and consistent amplification of HIC-1 with 10 % DMSO and 1.5 mM MgCl2. DMSO reduces the enzymatic activity of the DNA polymerase, so 1.5 times unit normally recommended was used. PCR with Taq polymerase generates product with 30 deoxyadenosine (dA) overhang and can directly clone into linearized plasmids with complementary single 50 deoxythymidine (dT) overhangs at both the ends. In our study, proofreading activity was required to obtain the exact protein sequence whereas polymerases with such activity do not produce 30 overhangs. PCR product can still be cloned into T-vector after a tailing procedure using Taq polymerase prior to ligation.

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A

M

B

1

1

M

C

3

4.7 kb

M

D

UD

10 kb

3 kbp 1843 bp

2 kbp

1843 bp

2 kb

1853 bp 1 kb

1 kbp

500 bp

Fig. 4 Cloning of F2R2 (large) fragment. a PCR with F2 and R2 primer gave band of 1,853 bp. (Lane M) marker, (Lane 1) PCR product. b Digestion of F2R2 and pPROEXHTb with restriction endonuclease (BamHI and SacI). (Lane 1) digested product of F2R2,

(Lane 3) back bone, (Lane M) is marker. c Digestion of pPROEXHTb with restriction endonuclease BamHI and SacI released an insert of 1,843 bb. M DNA marker, D digested, UD undigested plasmid

F2R2

UN

B

A

D

M

F1R1 D

UN

1825 bp

827 bp 450 bp 377 bp

C

Fig. 5 Full-length HIC-1 clone. a Gel-eluted products obtained from digestion of F1R1 and F2R2 cloned plasmid with BamHI and SacI. (Lane 1) F1R1 fragment, (Lane 3) F2R2, (Lane M) marker. b Digestion of F2R2 and F1R1with XhoII. M DNA marker, D digested, UD undigested fragment. c (Lane 1) Full-length clone

D

377 bp

1825 bp

of 2.2 kb, (Lane M) marker. d Schematic diagram representing cloned full-length exon-2 with respective BamHI and SacI sites inserted into the expression vector ‘‘pPROEXHTb’’. The fragments of 377 and 1,825 bp were obtained after digesting F1R1 (BamHI, XhoII) and F2R2 (XhoII, SacI), respectively

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1

2

3 HIC-1 (74 kDa)

Fig. 6 Protein expression of full-length HIC-1 in BL21-Codon PlusRP strain of E. coli. Lane 1 untransformed BL21-Codon Plus-RP strain, Lane 2 transformed and uninduced, Lane 3 transformed and IPTG induced. The respective coomassie staining is shown below

Once cloned, sequencing of high GC-rich products poses a daunting task. The presence of GC-rich sequences, di-, tri- and tetra-repeats and short tandem repeats in various genomes is problematic. DNA polymerase has been shown to pause or slip at the beginning of regions of strong secondary structures and gives base anomalies which arise due to secondary structures and intramolecular base pairing between guanine and cytosine bases. Betaine relieves the DNA polymerase pause near putative hairpin-loop structures of DNA sequences [15]. A common approach to sequence such reactions is to replace guanine with deoxyinositol triphosphate (dITP) [16] or 7-de aza dGTP [17] or to add denaturants such as DMSO or formamide or betaine to the reaction mixture to overcome sequencing artifacts in automated DNA sequencer with universal M13 primer [13]. In structural and functional studies of human protein, the production of sufficient amount of protein is frequent bottleneck. Initial standardization using pHAT10 as expression vector did not show any induction of desired fragments in E. coli DH5a with IPTG induction. The problem of codon bias has been most thoroughly documented for the arginine codons AGA and AGG, which are the rarest codons in E. coli [18]. However, codons for isoleucine (AUA), leucine (CUA), and proline (CCC) are also known to affect the amount and quality of protein produced in E. coli hosts. When the mRNA of heterologous target genes is overexpressed in E. coli, differences in codon usage can impede translation due to the demand for one or more tRNAs that may be rare or lacking in the tRNA population [18, 19]. BL21-Codon Plus-RIL contain extra copies of the argU, ileY, and leuW tRNA genes whereas the BL21-Codon Plus-RP strains contain the argU and proL tRNA genes. Unpublished report from our laboratory had confirmed that BL21-Codon Plus-RIL cells rescue expression of AT-rich genes and BL21-Codon Plus-RP cells rescue gene expression from GC-rich targets. Degradation of produced proteins is often caused by endogenous proteases whereas E. coli BL21 strain lacks both the Lon

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protease and ompT outer membrane protease [20]. Previously made pHAT clones were transformed to BL21Codon Plus-RP to overcome the problem of codon bias but none of the clones showed any noticeable induction of protein expression with IPTG. Initial failure to express in pHAT10, the cloned fragment were reamplified with another series of adapter primers designed as per expression vector pProEX-HTb, directionally cloned and transformed into E. coli DH5a as well as in BL21-Codon PlusRP. The lack of protein induction in pHAT10 clones was overcome by the pProEX-HTb clones. The pProEX-HTb clones in BL21-Codon Plus-RP showed proper induction with IPTG. The pProEX-HTb vector has a very strong Trc promoter as compared to the only Lac promoter in pHAT10 that might explain the high level of induction in pProEX-HTb clones. In current scenario, small peptide sequence expected having immunogenic properties of HIC-1 is used to generate antibody but the problem encountered by such synthetic peptides that they binds well to denatured proteins but may or may not recognize the native protein. Assays will succeed only when the peptide sequence is displayed on the surface of the native molecule in a conformation similar to the peptidecarrier conjugate. Therefore, the successful production of anti-peptide antibodies is often determined by the ability to predict the location of certain peptide sequences in the threedimensional structure of the protein. Biophysical studies of HIC-1 needs crystallization of full-length protein and later used for structural and functional studies, in addition to, homology search as well as to reveal interacting partners. It can also predict specificity of binding sequences while acting as transcription factor and tumor suppressor which is grossly lacking due to unavailability of full-length protein and can be resolved once having protein in hand. Acknowledgments This work was financially supported by Council of Scientific and Industrial Research (CSIR), Government of India for granting senior research fellowship. We thank Professor Subrata Sinha, Director, National Brain Research Centre, Manesar, Gurgaon for providing laboratory support to the work. We also thank Late Mathura Prasad, Jitender Behari Lal for technical support and Satish Kumar for secretarial assistance. Conflict of interest

None.

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