Abstract
Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Borgnia MJ, Kozono D, Calamita G, Maloney PC, Agre P (1999a) Functional Reconstitution and Characterization of AqpZ, the E. coli Water Channel Protein. J Mol Biol 291:1169–1179
Borgnia MJ, Nielsen M, Engel A, Agre P (1999b) Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 68:425–458
Bruno M, John EW (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298
Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G (2003) Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies. Protein Expres Purif 30:253–261
Calamita G (2000) The Escherichia coli aquaporin-Z water channel. Mol Microbiol 37(2):254–262
Calamita G, Bishai WR, Preston GM, Guggion WB, Agre P (1995) Molecular Cloning and Characerization of AqpZ, a Water Channel from Escherichia coli. J Biol Chem 270:29063–29066
Chen GQ, Gouaux JE (1996) Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin. Protein Sci 5:456–467
Douglas JL, Trieber CA, Afara M, Young HS (2005) Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies. Protein Expres Purif 40:118–125
Grisshammer R, Tate CG (1995) Overexpression of integral membrane proteins for structural studies. Q Rev Biophys 28:315–422
Grisshammer R, Little J, Aharony D (1994) Expression of rat NK-2 (neurokinin A) receptor in E. coli. Recept Channels 2:295–302
Huang L, Xu ZN, Zhong ZX, Peng L, Chen HQ, Cen PL (2007) Enhance expression and primary purification of soluble HBD3 fusion protein in Escherichia coli. Appl Biochem Biotechnol 142(2):139–147
Kiefer H, Krieger J, Olszewski JD, Heijne G, Prestwich GD, Breer H (1996) Expression of an Olfactory Receptor in Escherichia coli: purification, reconstitution, and ligand binding. Biochemistry 35:16077–16084
Kiefer H, Vogel R, Mailer K (2000) Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence. Recept Channels 7:109–119
Kozono D, Ding XD, Iwasaki I, Meng XY, Kamagata Y, Agre P, Kitagawa Y (2003) Functional expression and characterization of an Archaeal aquaporin: AqpM from Methanothermobacter marburgensis. J Biol Chem 278(12):10649–10656
Laage R, Langosch D (2001) Strategies for prokaryotic expression of eukaryotic membrane proteins. Traffic 2:99–104
LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 11:187–193
Lian JZ, Fang XM, Cai J, Chen QX, Zheng Q, Kai L, Xu ZN (2008) Efficient expression of membrane-bound water channel protein (Aquaporin Z) in Escherichia coli. Protein Pept Lett 15:687–691
Maduke M, Pheasant DJ, Miller C (1999) High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel. J Gen Physiol 114:713–722
Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60(3):512–538
Nygren PA, Stahl S, Uhlen M (1994) Engineering proteins to facilitate bioprocessing. Trends Biotechnol 12:184–188
Peng L, Xu ZN, Fang XM, Wang F, Cen PL (2004) High-level expression of soluble human Beta-Defensin-2 in E. coli. Process Biochem 39:2199–2205
Pompejus M, Friedrich K, Teufel M, Fritz HJ (1993) High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli. Eur J Biochem 211:27–35
Power RF, Conneely OM, McDonnell DP, Clark JH, Butt TR, Schrader WT, O’Malley BW (1990) High level expression of a truncated chicken progesterone receptor in Escherichia coli. J Biol Chem 265:1419–1424
Pryor KD, Leiting B (1997) High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. Protein Expres Purif 10:309–319
Samuelsson E, Moks T, Nilsson B, Uhlen M (1994) Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner. Biochemistry 33:4207–4211
Stanasila L, Massotte D, KieVer BL, Pattus F (1999) Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins. Eur J Biochem 260:430–438
Swartz JR (2006) Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol 33:476–485
Tate CG (2001) Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett 504(3):94–98
Voges D, Jap BK (1998) Recombinant expression, purification and characterization of Kch, a putative Escherichia coli potassium channel protein. FEBS Lett 429:104–108
Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD (2003) Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim Biophys Acta 1610:23–36
Xu ZN, Zhong ZX, Huang L, Peng L, Wang F, Cen PL (2006) High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl Microbiol Biotechnol 72:471–479
Yao Q, Bevan JL, Weaver RF, Bigelow DJ (1996) Purification of porcine phospholamban expressed in Escherichia coli. Protein Expres Purif 8:463–468
Zhang Y, Olsen DR, Nguyen KB, Olson PS, Rhodes ET, Mascarenhas D (1998) Expression of eukaryotic proteins in soluble form in Escherichia coli. Protein Expres Purif 12:159–165
Zhong ZX, Xu ZN, Peng L, Huang L, Fang XM, Cen PL (2006) Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli. Appl Microbiol Biotechnol 71(5):661–667
Acknowledgments
This work was financially supported by The National Natural Science Foundation of China (Grant No.20736008, 20676115), The Ministry of Science and Technology (Grant No 2007AA021702) and The Ministry of Education (Grant No. 20060335085), The People’s Republic of China.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lian, J., Ding, S., Cai, J. et al. Improving aquaporin Z expression in Escherichia coli by fusion partners and subsequent condition optimization. Appl Microbiol Biotechnol 82, 463–470 (2009). https://doi.org/10.1007/s00253-008-1774-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-008-1774-x