A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.
Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format.
Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708-0281, USA.
Miriam
M
Amiram
Felipe Garcia
FG
Quiroz
Daniel J
DJ
Callahan
Ashutosh
A
Chilkoti
D001704:Biopolymers / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry; D004549:Elastin / Q000737:chemistry; D015723:Gene Library; D005813:Genes, Synthetic; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D001704:Biopolymers; D004270:DNA, Circular; D011506:Proteins; D052216:Glucagon-Like Peptide 1; D004247:DNA; D004549:Elastin
Biopolymers. 2010;94(1):60-77
20091871
Chirality. 2008 Sep;20(9):985-94
18293367
Biomacromolecules. 2004 May-Jun;5(3):846-51
15132671
Protein Sci. 2009 Jul;18(7):1377-87
19533768
J Clin Microbiol. 2002 Jan;40(1):128-32
11773105
Gene. 1998 May 12;211(2):277-85
9602151
Int J Hyperthermia. 2008 Sep;24(6):483-95
18608590
J Am Chem Soc. 2008 Jan 16;130(2):687-94
18085778
Proc Natl Acad Sci U S A. 1995 May 9;92(10):4641-5
7753856
Mol Pathol. 2001 Oct;54(5):351-3
11577179
Biosci Biotechnol Biochem. 2007 Jun;71(6):1462-9
17587676
Biomacromolecules. 2006 Jul;7(7):2099-107
16827576
Gene. 1985;39(2-3):239-45
2419204
Nat Protoc. 2008;3(8):1312-7
18714299
Anal Biochem. 1991 Dec;199(2):184-90
1812783
Protein Eng Des Sel. 2007 Jan;20(1):25-32
17218334
Nature. 2010 May 6;465(7294):69-73
20445626
Biomacromolecules. 2003 Nov-Dec;4(6):1680-5
14606895
Biomacromolecules. 2002 Mar-Apr;3(2):357-67
11888323
Biotechnol Lett. 2005 May;27(9):665-70
15977075
Peptides. 2005 May;26(5):721-9
15808901
Tissue Eng Part A. 2008 Jul;14(7):1133-40
18433311
Diabetes. 2004 Sep;53(9):2492-500
15331566
Biotechnol Prog. 2004 Nov-Dec;20(6):1855-60
15575722
Appl Microbiol Biotechnol. 2002 May;58(6):790-6
12021800
Mater Sci Eng R Rep. 2008 Jan;62(4):125-155
19122836
Diabetes Obes Metab. 2009 Oct;11(10):953-65
19531053
Angew Chem Int Ed Engl. 2010 Mar 8;49(11):1971-5
20148425
Biomacromolecules. 2010 Apr 12;11(4):944-52
20184309
Adv Drug Deliv Rev. 2010 Dec 30;62(15):1404-55
20655344
Nat Methods. 2008 Jan;5(1):37-9
18037891
Appl Biochem Biotechnol. 2007 May-Jun;141(2-3):203-13
18025552
J Pept Sci. 2007 Jul;13(7):458-67
17559067
Angew Chem Int Ed Engl. 2010 Jul 5;49(29):4930-3
20540123