High quality protein microarray using in situ protein purification.
BACKGROUND
In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.


RESULTS
Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG) coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.


CONCLUSIONS
An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.
Kwon
Keehwan
K
Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA. kkwon@jcvi.org
Grose
Carissa
C
Pieper
Rembert
R
Pandya
Gagan A
GA
Fleischmann
Robert D
RD
Peterson
Scott N
SN
D001426:Bacterial Proteins / Q000302:isolation & purification; D002614:Chelating Agents; D003300:Copper; D004926:Escherichia coli / Q000235:genetics; D009532:Nickel; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013296:Streptococcus pneumoniae / Q000378:metabolism
D001426:Bacterial Proteins; D002614:Chelating Agents; D011994:Recombinant Proteins; D003300:Copper; D009532:Nickel
Nat Genet. 2002 Dec;32 Suppl:526-32
12454649
Nat Methods. 2008 Jan;5(1):69-74
18084297
Nature. 2006 Jan 12;439(7073):168-74
16273093
Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):547-52
15647345
J Bacteriol. 2006 Feb;188(3):1031-8
16428407
Proteomics. 2006 Aug;6(15):4227-34
16826567
Science. 2007 Jul 20;317(5836):364-9
17641200
Curr Opin Biotechnol. 2008 Feb;19(1):4-9
18207731
Biotechnol Adv. 2003 Nov;21(8):695-713
14563476
Nat Methods. 2008 Jun;5(6):535-8
18469824
Science. 2000 Sep 8;289(5485):1760-3
10976071
J Mol Biol. 1998 Oct 23;283(2):489-506
9769220
Eur Biophys J. 2006 Oct;35(8):655-62
16724194
Nat Struct Biol. 2000 Oct;7(10):903-9
11017201
Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2654-9
11880620
BMC Microbiol. 2007 Jun 25;7:59
17593301
Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
9783223
Anal Chem. 2006 May 1;78(9):3072-9
16642995
Infect Immun. 2001 Nov;69(11):6702-6
11598041
Nucleic Acids Res. 2001 Aug 1;29(15):E73-3
11470888
Protein Expr Purif. 2007 Oct;55(2):368-78
17703947
Protein Expr Purif. 2002 Jun;25(1):1-7
12071692
Methods Mol Biol. 2009;498:229-44
18988029
Langmuir. 2005 Aug 2;21(16):7071-5
16042424
Methods Mol Biol. 2009;498:31-54
18988017
Proteomics. 2007 Jun;7(11):1771-4
17476712
Nat Genet. 2000 Nov;26(3):283-9
11062466
Protein Expr Purif. 2001 Jul;22(2):159-64
11437590
Mol Cell Proteomics. 2006 Sep;5(9):1658-66
16825183
Curr Opin Chem Biol. 2001 Feb;5(1):40-5
11166646
Protein Expr Purif. 2004 Mar;34(1):49-55
14766299
Mol Microbiol. 2001 May;40(3):572-85
11359564
Proteomics. 2005 Feb;5(2):416-9
15627963
J Histochem Cytochem. 2003 Dec;51(12):1699-712
14623938
Science. 2004 Jul 2;305(5680):86-90
15232106
Mol Microbiol. 1998 Sep;29(5):1285-96
9767595
Anal Biochem. 2009 Sep 1;392(1):45-53
19464993
Proteomics. 2003 Mar;3(3):254-64
12627378
Bioinformatics. 1998;14(4):378-9
9632836
Infect Immun. 2004 Oct;72(10):5858-67
15385487
Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4011-6
16537477
Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11139-44
12167675
Science. 2001 Sep 14;293(5537):2101-5
11474067
Nat Methods. 2008 Jun;5(6):507-16
18511918
Expert Rev Proteomics. 2009 Apr;6(2):145-57
19385942
Proteomics. 2007 Jun;7(13):2172-83
17533643
Nat Biotechnol. 2000 Apr;18(4):393-7
10748518
Brief Funct Genomic Proteomic. 2004 Feb;2(4):308-19
15163366