Cold shock induction of recombinant Arctic environmental genes

BMC Biotechnology

Gro Elin Kjæreng Bjerga¹  Adele Kim Williamson² 

[1] Centre for Applied Biotechnology, Uni Research AS, Thormøhlensgt. 55, Bergen, N-5008, Norway

[2] Norstruct, Department of Chemistry, Faculty of Science and Technology, University of Tromsø, Tromsø, N-9037, Norway

关键词: Metagenomic DNA;    Cold-adapted;    Restriction-free cloning;    Fusion partner;    Heterologous expression;    cspA promoter;   

Others  :  1222671
DOI  :  10.1186/s12896-015-0185-1

 received in 2015-02-17, accepted in 2015-07-24,  发布年份 2015

【 摘 要 】

Background

Heterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry.

Methods

As part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags.

Results

We found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the low-temperature optimized vector suite.

Conclusions

We conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth.

【 授权许可】

   
2015 Bjerga and Williamson.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

【 参考文献 】

• [1]Demirjian DC, Moris-Varas F, Cassidy CS. Enzymes from extremophiles. Curr Opin Chem Biol. 2001; 5(2):144-51.
• [2]van den Burg B. Extremophiles as a source for novel enzymes. Curr Opin Microbiol. 2003; 6(3):213-8.
• [3]Ferrer M, Golyshina O, Beloqui A, Golyshin PN. Mining enzymes from extreme environments. Curr Opin Microbiol. 2007; 10(3):207-14.
• [4]Feller G. Psychrophilic enzymes: from folding to function and biotechnology. Scientifica. 2013; 2013:512840.
• [5]Feller G, Le Bussy O, Gerday C. Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol. 1998; 64(3):1163-5.
• [6]Smalas AO, Leiros HK, Os V, Willassen NP. Cold adapted enzymes. Biotechnol Annu Rev. 2000; 6:1-57.
• [7]Feller G. Life at low temperatures: is disorder the driving force? Extremophiles. 2007; 11(2):211-6.
• [8]Gerday C, Aittaleb M, Bentahir M, Chessa J-P, Claverie P, Collins T et al.. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 2000; 18(3):103-7.
• [9]Tanabe H, Goldstein J, Yang M, Inouye M. Identification of the promoter region of the Escherichia coli major cold shock gene, cspA. J Bacteriol. 1992; 174(12):3867-73.
• [10]Vasina JA, Baneyx F. Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Appl Environ Microbiol. 1996; 62(4):1444-7.
• [11]Bedouelle H, Duplay P. Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. Eur Jo Biochem. 1988; 171(3):541-9.
• [12]di Guan C, Li P, Riggs PD, Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 1988; 67(1):21-30.
• [13]Kapust RB, Waugh DS. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 1999; 8(8):1668-74.
• [14]Esaki K, Terashima Y, Toda E, Yoshinaga S, Araki N, Matsushima K et al.. Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5. Protein Expr Purif. 2011; 77(1):86-91.
• [15]Hoffmann A, Merz F, Rutkowska A, Zachmann-Brand B, Deuerling E, Bukau B. Trigger factor forms a protective shield for nascent polypeptides at the ribosome. J Biol Chem. 2006; 281(10):6539-45.
• [16]LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (Nature Publishing Company). 1993; 11(2):187-93.
• [17]Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics. 2004; 5(1–2):75-86.
• [18]Smith MC, Furman TC, Ingolia TD, Pidgeon C. Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins. J Biol Chem. 1988; 263(15):7211-5.
• [19]Hochuli E. Large-scale chromatography of recombinant proteins. J Chromatogr. 1988; 444:293-302.
• [20]CSteen J, Uhlen M, Hober S, Ottosson J. High-throughput protein purification using an automated set-up for high-yield affinity chromatography. Protein Expr Purif. 2006;46(2):173–8.
• [21]Schafer F, Romer U, Emmerlich M, Blumer J, Lubenow H, Steinert K. Automated high-throughput purification of 6xHis-tagged proteins. J Biomolec Tech. 2002; 13(3):131-42.
• [22]van den Ent F, Lowe J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods. 2006; 67(1):67-74.
• [23]Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol. 2010; 172(1):34-44.
• [24]Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM et al.. Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol. 2004; 22(7):877-82.
• [25]Fu J, Leiros HK, de Pascale D, Johnson KA, Blencke HM, Landfald B. Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library. Appl Microbiol Biotechnol. 2013; 97(9):3965-78.
• [26]Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA et al.. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005; 437(7057):376-80.
• [27]Zhu W, Lomsadze A, Borodovsky M. Ab initio gene identification in metagenomic sequences. Nucleic Acids Res. 2010; 38(12):e132.
• [28]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10.
• [29]Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011; 8(10):785-6.
• [30]Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR et al.. Pfam: the protein families database. Nucleic Acids Res. 2014; 42(D1):D222-30.
• [31]Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics (Oxford, England). 2006; 22(2):195-201.
• [32]Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD et al.. Protein identification and analysis tools in the ExPASy server. Methods Mole Biol (Clifton, NJ). 1999; 112:531-52.
• [33]Gräslund S, Sagemark J, Berglund H, Dahlgren L-G, Flores A, Hammarström M et al.. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr Purif. 2008; 58(2):210-21.
• [34]Lund BA, Leiros HK, Bjerga GE. A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement. Microb Cell Fact. 2014; 13(1):38. BioMed Central Full Text
• [35]Bond SR, Naus CC. RF-Cloning.org: an online tool for the design of restriction-free cloning projects. Nucleic Acids Res. 2012; 40(Web Server issue):W209-13.
• [36]Busso D, Delagoutte-Busso B, Moras D. Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli. Anal Biochem. 2005; 343(2):313-21.
• [37]Vincentelli R, Cimino A, Geerlof A, Kubo A, Satou Y, Cambillau C. High-throughput protein expression screening and purification in Escherichia coli. Methods. 2011; 55(1):65-72.
• [38]Broeze RJ, Solomon CJ, Pope DH. Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens. J Bacteriol. 1978; 134(3):861-74.
• [39]Shaw MK, Ingraham JL. Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth. J Bacteriol. 1967; 94(1):157-64.
• [40]Goldenberg D, Azar I, Oppenheim AB. Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli. Mol Microbiol. 1996; 19(2):241-8.
• [41]Brandi A, Pietroni P, Gualerzi CO, Pon CL. Post-transcriptional regulation of CspA expression in Escherichia coli. Mol Microbiol. 1996; 19(2):231-40.
• [42]Mitta M, Fang L, Inouye M. Deletion analysis of cspA of Escherichia coli: requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction. Mol Microbiol. 1997; 26(2):321-35.
• [43]Fang L, Jiang W, Bae W, Inouye M. Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization. Mol Microbiol. 1997; 23(2):355-64.
• [44]Goldstein J, Pollitt NS, Inouye M. Major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A. 1990; 87(1):283-7.
• [45]Xia B, Etchegaray JP, Inouye M. Nonsense mutations in cspA cause ribosome trapping leading to complete growth inhibition and cell death at low temperature in Escherichia coli. J Biol Chem. 2001; 276(38):35581-8.
• [46]Jiang W, Fang L, Inouye M. Complete growth inhibition of Escherichia coli by ribosome trapping with truncated cspA mRNA at low temperature. Genes Cells. 1996; 1(11):965-76.
• [47]Rosano GL, Ceccarelli EA. Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Fact. 2009; 8:41. BioMed Central Full Text
• [48]Goldman E, Rosenberg AH, Zubay G, Studier FW. Consecutive low-usage leucine codons block translation only when near the 5′ end of a message in Escherichia coli. J Mol Biol. 1995; 245(5):467-73.
• [49]Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol. 1995; 6(5):494-500.
• [50]Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN. Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol. 2003; 21(11):1266-7.
• [51]Ueda M, Ito A, Nakazawa M, Miyatake K, Sakaguchi M, Inouye K. Cloning and expression of the cold-adapted endo-1,4-beta-glucanase gene from Eisenia fetida. Carbohydr Polym. 2014; 101:511-6.
• [52]Hayashi K, Kojima C. pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production. Protein Expr Purif. 2008; 62(1):120-7.
• [53]Hayashi K, Kojima C. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector. J Biomol NMR. 2010; 48(3):147-55.
• [54]Bird LE. High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli. Methods. 2011; 55(1):29-37.
• [55]Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 2006; 15(1):182-9.
• [56]Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard T. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 2002; 11(2):313-21.
• [57]Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P. Generation of a vector suite for protein solubility screening. Front Microbiol. 2014; 5:67.
• [58]Niiranen L, Espelid S, Karlsen CR, Mustonen M, Paulsen SM, Heikinheimo P et al.. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. Protein Expr Purif. 2007; 52(1):210-8.
• [59]Shih YP, Kung WM, Chen JC, Yeh CH, Wang AH, Wang TF. High-throughput screening of soluble recombinant proteins. Protein Sci. 2002; 11(7):1714-9.
• [60]Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014; 42(Database issue):D490-5.
• [61]Henrissat B, Davies G. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997; 7(5):637-44.
• [62]Howard MB, Ekborg NA, Taylor LE, Weiner RM, Hutcheson SW. Chitinase B of “Microbulbifer degradans” 2–40 contains two catalytic domains with different chitinolytic activities. J Bacteriol. 2004; 186(5):1297-303.
• [63]Horn SJ, Sørlie M, Vaaje-Kolstad G, Norberg AL, Synstad B, Vårum KM et al.. Comparative studies of chitinases A, B and C from Serratia marcescens. Biocatal Biotransform. 2006; 24(1–2):39-53.
• [64]Lonhienne T, Mavromatis K, Vorgias CE, Buchon L, Gerday C, Bouriotis V. Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes. J Bacteriol. 2001; 183(5):1773-9.
• [65]Hult EL, Katouno F, Uchiyama T, Watanabe T, Sugiyama J. Molecular directionality in crystalline beta-chitin: hydrolysis by chitinases A and B from Serratia marcescens 2170. Biochem J. 2005; 388(Pt 3):851-6.
• [66]Goni O, Sanchez-Ballesta MT, Merodio C, Escribano MI. Two cold-induced family 19 glycosyl hydrolases from cherimoya (Annona cherimola) fruit: an antifungal chitinase and a cold-adapted chitinase. Phytochemistry. 2013; 95:94-104.
• [67]Ramli AN, Mahadi NM, Rabu A, Murad AM, Bakar FD, Illias RM. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12. Microb Cell Fact. 2011; 10:94. BioMed Central Full Text
• [68]Orikoshi H, Baba N, Nakayama S, Kashu H, Miyamoto K, Yasuda M et al.. Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. strain O-7. J Bacteriol. 2003; 185(4):1153-60.
• [69]Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H, Uchida M et al.. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem. 1993; 268(25):18567-72.
• [70]van Aalten DM, Komander D, Synstad B, Gaseidnes S, Peter MG, Eijsink VG. Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci U S A. 2001; 98(16):8979-84.
• [71]Vaaje-Kolstad G, Houston DR, Rao FV, Peter MG, Synstad B, van Aalten DM et al.. Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin. Biochim Biophys Acta. 2004; 1696(1):103-11.
• [72]Kolstad G, Synstad B, Eijsink VG, van Aalten DM. Structure of the D140N mutant of chitinase B from Serratia marcescens at 1.45 A resolution. Acta Crystallogr D Biol Crystallogr. 2002; 58(Pt 2):377-9.
• [73]Synstad B, Gaseidnes S, Van Aalten DM, Vriend G, Nielsen JE, Eijsink VG. Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase. Eur J Biochem. 2004; 271(2):253-62.
• [74]Tronsmo A, Harman GE. Detection and quantification of N-acetyl-beta-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal Biochem. 1993; 208(1):74-9.
• [75]Butt TR, Edavettal SC, Hall JP, Mattern MR. SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif. 2005; 43(1):1-9.
• [76]Nallamsetty S, Waugh DS. Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr Purif. 2006; 45(1):175-82.
• [77]Chant A, Kraemer-Pecore CM, Watkin R, Kneale GG. Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expr Purif. 2005; 39(2):152-9.
• [78]Williamson A, Pedersen H. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida. Protein Expr Purif. 2014; 97:29-36.
• [79]Chen X, Zaro JL, Shen W-C. Fusion protein linkers: Property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69.
• [80]Gromek KA, Meddaugh HR, Wrobel RL, Suchy FP, Bingman CA, Primm JG et al.. Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence. Protein Expr Purif. 2013; 89(2):203-9.
• [81]Smyth DR, Mrozkiewicz MK, McGrath WJ, Listwan P, Kobe B. Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 2003; 12(7):1313-22.
• [82]Arai R, Wriggers W, Nishikawa Y, Nagamune T, Fujisawa T. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. Proteins. 2004; 57(4):829-38.
• [83]Tamura M, Ito K, Kunihiro S, Yamasaki C, Haragauchi M. Production of human beta-actin and a mutant using a bacterial expression system with a cold shock vector. Protein Expr Purif. 2011; 78(1):1-5.
• [84]Kim EK, Moon JC, Lee JM, Jeong MS, Oh C, Ahn SM et al.. Large-scale production of soluble recombinant amyloid-beta peptide 1–42 using cold-inducible expression system. Protein Expr Purif. 2012; 86(1):53-7.
• [85]Mujacic M, Cooper KW, Baneyx F. Cold-inducible cloning vectors for low-temperature protein expression in Escherichia coli: application to the production of a toxic and proteolytically sensitive fusion protein. Gene. 1999; 238(2):325-32.