title: Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification.
abstract: The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.
authors: Johns Hopkins University, Department of Chemical and Biomolecular Engineering, 3400 North Charles Street, Baltimore, MD 21218, United States. Electronic address: twarre12@jhu.edu.
authors: Tiana D
authors: TD
authors: Warren
authors: Michael J
authors: MJ
authors: Coolbaugh
authors: David W
authors: DW
authors: Wood
mesh_terms: D001483:Base Sequence; D002686:Chitin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057166:High-Throughput Screening Assays; D047668:Inteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D002686:Chitin
keywords: Intein; Ligation-independent cloning; Molecular cloning; Protein purification; Self-cleaving affinity tag
references: Protein Expr Purif. 2007 May;53(1):40-50
references: 17251035
references: J Struct Biol. 2011 Aug;175(2):113-9
references: 21453775
references: J Biol Chem. 1996 Sep 6;271(36):22159-68
references: 8703028
references: J Biol Chem. 1998 Apr 24;273(17):10567-77
references: 9553117
references: Annu Rev Microbiol. 2002;56:263-87
references: 12142479
references: Nucleic Acids Res. 1990 Oct 25;18(20):6069-74
references: 2235490
references: Protein Expr Purif. 2002 Jun;25(1):1-7
references: 12071692
references: Nat Methods. 2005 Sep;2(9):659-61
references: 16074986
references: J Biol Chem. 1994 Dec 23;269(51):32678-84
references: 7798275
references: J Bacteriol. 1991 Sep;173(18):5653-62
references: 1909321
references: Nucleic Acids Res. 1997 Oct 15;25(20):4165-6
references: 9321675
references: Trends Biotechnol. 2010 May;28(5):272-9
references: 20359761
references: Gene. 1999 Apr 29;231(1-2):1-13
references: 10231563
references: Biotechnol Prog. 2000 Nov-Dec;16(6):1055-63
references: 11101334
references: PCR Methods Appl. 1994 Dec;4(3):172-7
references: 7580902
references: Appl Microbiol Biotechnol. 2012 Mar;93(5):1853-63
references: 22311648
references: Protein Expr Purif. 2009 Aug;66(2):198-202
references: 19345265
references: Biotechniques. 1999 Jul;27(1):110-4, 116, 118-20
references: 10407673
references: BMC Biotechnol. 2006 Mar 01;6:12
references: 16509985
references: Protein Expr Purif. 2002 Jun;25(1):8-15
references: 12071693
references: Curr Protoc Protein Sci. 2009 Nov;Chapter 26:Unit 26.4.1-18
references: 19937722
references: Biotechnol Prog. 2010 Sep-Oct;26(5):1205-12
references: 20945478
references: Genome Res. 2000 Nov;10(11):1788-95
references: 11076863
references: BMC Biotechnol. 2008 Mar 27;8:32
references: 18371187
references: Biotechnol Bioeng. 2008 Oct 1;101(2):229-40
references: 18727029
references: Nat Biotechnol. 1999 Sep;17(9):889-92
references: 10471931