The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection.

[1]  K. Shinozaki,et al.  The effect of overexpression of two Brassica CBF/DREB1-like transcription factors on photosynthetic capacity and freezing tolerance in Brassica napus. , 2005, Plant & cell physiology.

[2]  S. Sim,et al.  Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coli protein, RpoS. , 2008 .

[3]  Joël Janin,et al.  Refolding strategies from inclusion bodies in a structural genomics project , 2004, Journal of Structural and Functional Genomics.

[4]  M. Thomashow,et al.  DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide. , 1992, Plant physiology.

[5]  R. Herrmann,et al.  The single‐copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II , 1992, FEBS letters.

[6]  A. Tunnacliffe,et al.  LEA proteins prevent protein aggregation due to water stress. , 2005, The Biochemical journal.

[7]  J. Mccoy,et al.  A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm , 1993, Bio/Technology.

[8]  A. Ohta,et al.  A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae. , 1996, Gene.

[9]  Susan Idicula-Thomas,et al.  Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli , 2005, Protein science : a publication of the Protein Society.

[10]  T. Kakizaki,et al.  Evaluation of the Protective Activities of a Late Embryogenesis Abundant (LEA) Related Protein, Cor15am, during Various Stresses in Vitro , 2008, Bioscience, biotechnology, and biochemistry.

[11]  C. Tate,et al.  Overexpression of mammalian integral membrane proteins for structural studies , 2001, FEBS letters.

[12]  R G Harrison,et al.  New fusion protein systems designed to give soluble expression in Escherichia coli. , 1999, Biotechnology and bioengineering.

[13]  H. Kamada,et al.  Arabidopsis thaliana ECP63 encoding a LEA protein is located in chromosome 4. , 1997, Gene.

[14]  E. Bergantino,et al.  Light- and pH-dependent structural changes in the PsbS subunit of photosystem II , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  John L Markley,et al.  Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress‐related protein , 2005, Protein science : a publication of the Protein Society.

[16]  D. Maeder,et al.  Isolation and characterization of a heat-soluble protein from pea (Pisum sativum) embryos , 1995, Seed Science Research.

[17]  A. Tunnacliffe,et al.  Anhydrobiosis: Plant desiccation gene found in a nematode , 2002, Nature.

[18]  M. Wise,et al.  The continuing conundrum of the LEA proteins , 2007, Naturwissenschaften.

[19]  D. Waugh,et al.  Single amino acid substitutions on the surface of Escherichia coli maltose‐binding protein can have a profound impact on the solubility of fusion proteins , 2001, Protein science : a publication of the Protein Society.

[20]  R. Peterson,et al.  Phylogeny-directed structural analysis of the Arabidopsis PsbS protein. , 2007, Biochemical and biophysical research communications.

[21]  G. Montelione,et al.  Enhancement of the solubility of proteins overexpressed in Escherichia coli by heat shock. , 2002, Journal of molecular microbiology and biotechnology.

[22]  K. Wakui,et al.  Isolation and expression of Lea gene in desiccation-tolerant microspore-derived embryos in Brassica spp. , 2002, Physiologia plantarum.

[23]  D. Waugh,et al.  Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused , 1999, Protein science : a publication of the Protein Society.

[24]  B. Leiting,et al.  High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. , 1997, Protein expression and purification.

[25]  D. Rubinsztein,et al.  Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function , 2007, Proceedings of the National Academy of Sciences.

[26]  T. Close,et al.  Cold-Specific Induction of a Dehydrin Gene Family Member in Barley , 1995, Plant physiology.

[27]  Dominic Esposito,et al.  Enhancement of soluble protein expression through the use of fusion tags. , 2006, Current opinion in biotechnology.

[28]  J. Cushman,et al.  Temperature-Induced Extended Helix/Random Coil Transitions in a Group 1 Late Embryogenesis-Abundant Protein from Soybean1 , 2002, Plant Physiology.

[29]  H. Kalbitzer,et al.  The recombinant dehydrin-like desiccation stress protein from the resurrection plant Craterostigma plantagineum displays no defined three-dimensional structure in its native state. , 1996, Biological chemistry.

[30]  G. Galau,et al.  Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. , 1981, Biochemistry.

[31]  C Marque,et al.  Human abdominal EHG processing for uterine contraction monitoring. , 1989, Biotechnology.

[32]  Michael F. Thomashow,et al.  PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms. , 1999, Annual review of plant physiology and plant molecular biology.

[33]  S. McCorkle,et al.  Protein aggregation during overexpression limited by peptide extensions with large net negative charge. , 2004, Protein expression and purification.

[34]  Shuying Feng,et al.  The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli. , 2007, Journal of biotechnology.

[35]  Michael J. Wise,et al.  LEAping to conclusions: A computational reanalysis of late embryogenesis abundant proteins and their possible roles , 2003, BMC Bioinformatics.

[36]  P. Karki,et al.  Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins. , 2008, BioTechniques.

[37]  T. Ho,et al.  Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice , 1996, Plant physiology.

[38]  H. Itoh,et al.  Arabidopsis Cor15am Is a Chloroplast Stromal Protein That Has Cryoprotective Activity and Forms Oligomers1[W][OA] , 2007, Plant Physiology.

[39]  E. Bray.  Classification of genes differentially expressed during water-deficit stress in Arabidopsis thaliana: an analysis using microarray and differential expression data. , 2002, Annals of botany.

[40]  Eriola Betiku,et al.  Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. , 2007, Journal of biotechnology.

[41]  M. Wise,et al.  POPP the question: what do LEA proteins do? , 2004, Trends in plant science.

[42]  U. Rinas,et al.  Kinetics of Heat‐Shock Response and Inclusion Body Formation During Temperature‐Induced Production of Basic Fibroblast Growth Factor in High‐Cell‐Density Cultures of Recombinant Escherichiacoli , 2000, Biotechnology progress.

[43]  P. Zhou,et al.  Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli. , 2008, Journal of biotechnology.

[44]  S. J. Gilmour,et al.  Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Zurdo,et al.  Transition from Natively Unfolded to Folded State Induced by Desiccation in an Anhydrobiotic Nematode Protein* , 2003, The Journal of Biological Chemistry.

[46]  P. Nordlund,et al.  An efficient strategy for high‐throughput expression screening of recombinant integral membrane proteins , 2005, Protein science : a publication of the Protein Society.

[47]  Chu di Guana,et al.  Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. , 1988 .

[48]  David L. Wilkinson,et al.  Predicting the Solubility of Recombinant Proteins in Escherichia coli , 1991, Bio/Technology.

[49]  S. J. Gilmour,et al.  Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[50]  T. White,et al.  Characterization of Three Related Low-Temperature-Regulated cDNAs from Winter Brassica napus , 1993, Plant physiology.

[51]  I. Glazer,et al.  An LEA group 3 family member is involved in survival of C. elegans during exposure to stress , 2004, FEBS letters.

[52]  J. Ingram,et al.  THE MOLECULAR BASIS OF DEHYDRATION TOLERANCE IN PLANTS. , 1996, Annual review of plant physiology and plant molecular biology.

[53]  M. Jaquinod,et al.  Structure and Function of a Mitochondrial Late Embryogenesis Abundant Protein Are Revealed by Desiccation[W] , 2007, The Plant Cell Online.

[54]  S. Goenka,et al.  Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification. , 2001, Protein expression and purification.

[55]  E. Nudler,et al.  Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.