doi:10.1016/j.pep.2006.12.017
Copyright © 2006 Elsevier Inc. All rights reserved.
Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: Preparation for a functional analysis
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Ahmed J. Afzala and David A. Lightfootb, 
, 
aDepartment of Molecular Biology, Microbiology and Biochemistry and Center for Excellence in Soybean Research, Teaching and Outreach, Southern Illinois University at Carbondale, IL 62901, USA
bGenomics Core Facility, Department of Plant Soil and Agricultural Systems, and Center for Excellence in Soybean Research, Teaching and Outreach, Southern Illinois University at Carbondale, Carbondale, IL 62901-4415, USA
Received 27 November 2006;
revised 19 December 2006.
Available online 27 December 2006.
Abstract
Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0–7.0 M urea in 0.5 M increments. Purified RHG1 protein obtained from the 1.5 and 7.0 M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0 M urea sample, whereas refolding from the 1.5 M urea sample yielded 0.2 mg/ml protein. The 7.0 M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.
Keywords: Plant disease resistance; SCN; Expression in E. coli; His-tag purification; Protein folding
Fig. 1. Domain architecture of complete RHG1 using the SMART (simple modular architecture research tool). Predicted signal peptide is shown as a dotted line (1–48), first transmembrane domain is shown as black rectangle (40–60), second single pass transmembrane domain shown as grey rectangle, serine threonine kinase domain (STYKc) (569–840) and 10 leucine-rich repeats are also shown (141–409). Arrows show the cloned LRR region (141–485).
Fig. 2. RLLH and RLSH cloning strategies. The rhg1 cDNA (Insert) was PCR amplified from a cDNA clone using NcoI and XhoI encoding primers. After amplification of template DNA, the PCR product was digested and cloned into the expression vector (pET30A). To generate RLSH, a 111 bp linker region between the 6-His encoding sequence and normal rhg1 start codon was deleted using mutagenesis with primer pair DMF1.
Fig. 3. A 12.5% (w/v) SDS–PAGE showing RHG1-LRR after induction as RLLH and RLSH. Lane 1, Benchmark protein ladder; lane 2, crude lysate of uninduced RLSH; lane 3, crude lysate showing induced RLSH; lane 4, crude lysate of uninduced RLLH; lane 5, crude lysate showing induced RLLH. Protein marker sizes were shown in kDa. The black arrow showed the position expected for RLLH and the white arrow showed the position of RLSH.
Fig. 4. A 12.5% (w/v) SDS–PAGE analysis of recombinant RHG1-LRR proteins. Lane 1, protein ladder; lane 2, soluble fraction from cell lysate of induced RLSH; lane 3, induced RLSH inclusion body lysate in 7.0 M urea (white arrow); lane 4, soluble fraction of induced cell lysate of RLLH; lane 5, induced RLLH inclusion body lysate in 7.0 M urea (black arrow). Protein marker sizes are shown in kDa.
Fig. 5. Urea titrations of RLLH eluted from RHG1-LRR IBs electrophoresed on 12.5% (w/v) SDS–PAGE. The RHG1-LRR inclusion body fraction was eluted with urea at; lane 1, 1.0 M; lane 2, 1.5 M; lane 3, 2.0 M; lane 4, 3.0 M; lane 5, 3.5 M; lane 7, 4.0 M; lane 8, 4.5 M; lane 9, 7.0 M urea. Lane 6 contained benchmark protein ladder. The sizes of the polypeptides were shown in kDa. Black arrows indicated the expected positions for the RHG1-LRR protein.
Fig. 6. A 12.5% (w/v) SDS–PAGE showing purified RHG1-LRR (both short and long his-tag) after elution at 1.5 M urea and affinity chromatography. Lane 1, refolded RLLH in pH 6.0 phosphate buffer; lane 2, refolded RLSH in pH 6.0 phosphate buffer; lane 3, protein marker. Protein marker sizes were shown in kDa.
Fig. 7. Western hybridization to RHG1 using an anti-His antibody. Lane 1, anti-His Western ladder; lane 2, RLLH; lane 3, RLSH. Protein marker sizes were shown in kDa.
Fig. 8. Circular dichroism of RLLH-native form of the RHG1-LRR in 20 mM phosphate buffer pH 6.0 (black-solid line), RLLH in 20 mM phosphate buffer containing 1.5 M urea ( gray-solid line) and RLLH in 20 mM phosphate buffer containing 7.0 M urea (black dotted line). The CD data was processed using an integrated software package termed CDTOOL.
Table 1.
Codon usage bias of RHG1-LRR in E. coli
Frequencies of rare arginine, leucine, isoleucine and proline codons in E. coli are shown for comparison.
Table 2.
Parameters determining protein stability and aggregation propensity of RHG1-LRR
Instability index and half life (* in yeast, # in bacteria) parameters were calculated from the webtool: Protparam found at http://us.expasy.org/cgi-bin/protparam. Protein contact order was calculated using a PDB file generated from ab initio model of RHG1-LRR (unpublished data) using the algorithm developed at the University of Washington: http://depts.washington.edu/bakerpg/contact_order/ and propensity of aggregation for peptides in RHG1-LRR was measured using the web-based algorithm TANGO: http://tango.embl.de/tangosecure/servlet/SecurePages?page=Calculation, predicted β sheet content was determined by CD data analysis of native-RHG1 using CDSSTR software (http://www.cryst.bbk.ac.uk/cdweb/html/home.html) and the folding was determined from the website: http://psfs.cbrc.jp/fold-rate/.
Table 3.
Secondary structure content of purified RHG1-LRR in the presence and absence of urea
Secondary structure was determined from web-tool: CDSSTR located at the dichroweb server (http://www.cryst.bbk.ac.uk/cdweb/html/home.html).
Corresponding author. Fax: +1 618 453 7457.
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