Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction.
Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 A resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.
Emamzadah
Soheila
S
Department of Molecular Biology, University of Geneva, Geneva, Switzerland.
Petty
Tom J
TJ
De Almeida
Victor
V
Nishimura
Taisuke
T
Joly
Jacques
J
Ferrer
Jean Luc
JL
Halazonetis
Thanos D
TD
D000074183:ATPases Associated with Diverse Cellular Activities; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003460:Crystallization; D003516:Cycloparaffins / Q000737:chemistry* / Q000378:metabolism; D023281:Genomics; D006801:Humans; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction
D029681:Arabidopsis Proteins; D003516:Cycloparaffins; D009687:Nuclear Proteins; D011108:Polymers; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D009113:Muramidase; D014357:Trypsin; D000074183:ATPases Associated with Diverse Cellular Activities; C409208:MOM protein, Arabidopsis
Nat Methods. 2008 Feb;5(2):147-53
18235435
Nature. 2000 May 11;405(6783):203-6
10821279
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
EMBO Rep. 2006 Dec;7(12):1279-84
17082818
J Struct Biol. 2003 Apr;142(1):218-31
12718933
J Am Chem Soc. 2003 Sep 17;125(37):11170-1
16220918
Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16531-6
12486223
Science. 1994 Jul 15;265(5170):346-55
8023157
Acta Crystallogr D Biol Crystallogr. 2001 Aug;57(Pt 8):1119-26
11468395
Science. 2006 Jan 20;311(5759):347-51
16424331
Proc Natl Acad Sci U S A. 2004 Oct 5;101(40):14431-6
15452343
Angew Chem Int Ed Engl. 2004 May 3;43(19):2508-11
15127437
Curr Opin Struct Biol. 2003 Oct;13(5):538-44
14568607
Science. 1991 Jul 5;253(5015):49-53
1905840
Acta Crystallogr D Biol Crystallogr. 2002 May;58(Pt 5):805-14
11976492
Acta Crystallogr D Biol Crystallogr. 2008 Feb;64(Pt 2):189-97
18219119
PLoS Genet. 2008 Aug 22;4(8):e1000165
18725928
Science. 2002 Nov 1;298(5595):948-50
12411682
Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
2025413
Lab Chip. 2006 Oct;6(10):1346-54
17102848
J Mol Biol. 2001 Mar 23;307(2):605-17
11254385
J Struct Biol. 2003 Apr;142(1):154-61
12718927
Science. 1995 Mar 10;267(5203):1498-502
7878469
Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19243-8
17159147
J Am Chem Soc. 2006 Mar 15;128(10):3142-3
16522084
Structure. 2004 Jul;12(7):1219-25
15242598