High-yield production, refolding and a molecular modelling of the catalytic module of (1,3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp.
Biosynthesis of the (1,3)-beta-D: -glucan (curdlan) in Agrobacterium sp., is believed to proceed by the repetitive addition of glucosyl residues from UDP-glucose by a membrane-embedded curdlan synthase (CrdS) [UDP-glucose: (1,3)-beta-D: -glucan 3-beta-D: -glucosyltransferase; EC 2.4.1.34]. The catalytic module of CrdS (cm-CrdS) was expressed in good yield from a cDNA encoding cm-CrdS cloned into the pET-32a(+) vector, containing a coding region for thioredoxin, and from the Champion pET SUMO system that possesses a coding region of a small ubiquitin-related modifier (SUMO) partner protein. The two DNA fusions, designated pET-32a_cm-CrdS and SUMO_cm-CrdS were expressed as chimeric proteins. High yields of inclusion bodies were produced in E. coli and these could be refolded to form soluble proteins, using a range of buffers and non-detergent sulfobetaines. A purification protocol was developed, which afforded a one-step on-column refolding and simultaneous purification of the recombinant 6xHis-tagged SUMO_cm-CrdS protein. The latter protein was digested by a specific protease to yield intact cm-CrdS in high yields. The refolded SUMO_cm-CrdS protein did not exhibit curdlan synthase activity, but showed a circular dischroism spectrum, which had an alpha/beta-type-like conformation. Amino acid sequences of tryptic fragments of the SUMO_cm-CrdS fusion and free cm-CrdS proteins, determined by MALDI/TOF confirmed that the full-length proteins were synthesized by E. coli, and that no alterations in amino acid sequences occurred. A three-dimensional model of cm-CrdS predicted the juxtaposition of highly conserved aspartates D156, D208, D210 and D304, and the QRTRW motif, which are likely to play roles in donor and acceptor substrate binding and catalysis.
Hrmova
Maria
M
Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA, 5064, Australia. maria.hrmova@adelaide.edu.au
Stone
Bruce A
BA
Fincher
Geoffrey B
GB
D020816:Amino Acid Motifs; D000818:Animals; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics
D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase
J Mol Biol. 1982 May 5;157(1):105-32
7108955
Protein Expr Purif. 2005 Aug;42(2):268-77
15914031
Nat Biotechnol. 1996 Mar;14(3):329-34
9630895
Plant Physiol. 1997 Oct;115(2):643-656
12223833
Biomed Chromatogr. 2007 Dec;21(12):1291-6
17634960
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Oct 1;62(Pt 10):1031-3
17012805
EMBO J. 1998 Oct 1;17(19):5543-50
9755155
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Aug 1;65(Pt 8):784-7
19652339
Planta. 1981 Jul;152(4):346-51
24301030
Glycobiology. 1999 Jan;9(1):31-41
9884404
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13431-6
9391042
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W526-31
15215442
Nature. 1970 Aug 15;227(5259):680-5
5432063
Protein Sci. 2004 Oct;13(10):2782-92
15388864
Biotechnol Bioeng. 2004 Mar 20;85(6):601-9
14966801
New Phytol. 2008;178(2):239-252
18298430
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
J Mol Biol. 1993 Dec 5;234(3):779-815
8254673
Protein Expr Purif. 2007 Apr;52(2):348-55
17166740
J Mol Biol. 2003 Apr 25;328(2):307-17
12691742
Glycobiology. 2003 Oct;13(10):693-706
12851288
Plant J. 2007 Oct;52(1):147-56
17666022
J Mol Biol. 1992 May 20;225(2):487-94
1593632
Bioinformatics. 2003 May 22;19(8):1015-8
12761065
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Apr 1;65(Pt 4):346-9
19342776
Plant Mol Biol. 2003 Sep;53(1-2):213-25
14756318
J Phys Chem B. 2008 Oct 23;112(42):13349-54
18816091
Bioinformatics. 2001 Sep;17(9):849-50
11590105
J Bacteriol. 2000 Sep;182(18):5029-35
10960084
Biochemistry. 1999 May 18;38(20):6380-5
10350455
Protein Eng Des Sel. 2004 Apr;17(4):305-14
15166312
Protein Eng. 1997 Jun;10(6):673-6
9278280
Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
18838391
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3318-21
6222377
Biochem J. 2008 Aug 15;414(1):43-52
18462191
Protein Sci. 1999 Jul;8(7):1475-83
10422836
J Struct Funct Genomics. 2005;6(2-3):103-11
16211506
Protein Expr Purif. 2000 Dec;20(3):379-88
11087677
Biochemistry. 2003 May 27;42(20):6264-74
12755631
Protein Expr Purif. 2007 Sep;55(1):166-74
17533138
Annu Rev Microbiol. 1992;46:307-46
1444258
Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
11517324
J Struct Funct Genomics. 2004;5(3):195-204
15263835
EMBO J. 2009 Sep 2;28(17):2677-85
19629046
Biochemistry. 1994 Mar 15;33(10):3038-49
8130217
J Bacteriol. 2002 Aug;184(15):4114-23
12107128
Protein Expr Purif. 2005 Sep;43(1):1-9
16084395
Proteins. 1993 Dec;17(4):355-62
8108378
J Mol Biol. 2001 Jan 19;305(3):567-80
11152613
Biochem Biophys Res Commun. 2005 Mar 25;328(4):1216-23
15708006
Bioinformatics. 2007 Apr 1;23(7):802-8
17267437
J Mol Biol. 1981 Mar 25;147(1):195-7
7265238
Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W177-81
16844986
J Struct Funct Genomics. 2006 Jun;7(2):77-91
17120110
J Struct Funct Genomics. 2004;5(1-2):75-86
15263846
Methods Enzymol. 1996;266:525-39
8743704
Cell Mol Life Sci. 1997 Aug;53(8):621-45
9351466
Protein Sci. 2005 Jul;14(7):1818-26
15937284
Biotechnol Appl Biochem. 2006 Mar;43(Pt 3):137-45
16293104
Protein Eng. 1999 May;12(5):381-5
10360978
Appl Microbiol Biotechnol. 2005 Aug;68(2):163-73
15818477
FEBS J. 2009 Jan;276(2):437-56
19076217
Biochem Biophys Res Commun. 2009 Jan 2;378(1):10-4
18771653
Cell Stress Chaperones. 2005 Winter;10(4):329-39
16333986