Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Klock, Heath E.; Koesema, Eric; Knuth, Mark W.; Lesley, Scott A.

doi:10.1002/prot.21786

Cited by 274 publications

(277 citation statements)

References 28 publications

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“…The cDNA encoding murine RBP4 (amino acid 19 -201) (Genomics Institute of the Novartis Research Foundation) was amplified with PCR using 2 primers designed specifically for the Polymerase Incomplete Primer Extension (PIPE) cloning method (27): 5Ј-CTGTACTTCCAGGGCGAGCGCGACTGCAGGG (5Ј insert forward primer) and 5Ј-AATTAAGTCGCGTTACAAACTGTTTCTGGAGGGCC (3Ј insert reverse primer). The pSpeedET vector was amplified using a 5Ј vector reverse primer 5Ј-GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGATG and a 3Ј vector forward primer 5Ј-TAACGCGACTTAATTAACTCGTTTA-AACGGTCTCCAGC.…”

Section: Construction Of Mrbp4 Expressionmentioning

confidence: 99%

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Grünewald¹,

Hunt²,

Dong

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269 -277]. However, the decreased ability of the immune system to mount a robust immune response to selfantigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO 2Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-␣ (mTNF-␣) independently to pNO2Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-␣ IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-␣ and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO 2Phe 43 mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.retinol-binding protein ͉ tumor necrosis factor ͉ vaccination ͉ p-nitrophenylalanine ͉ genetic code

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Section: Construction Of Mrbp4 Expressionmentioning

confidence: 99%

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Grünewald¹,

Hunt²,

Dong

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Many other methods have been developed for this purpose: Gateway cloning (Life Technologies), In-Fusion cloning (TAKARA BIO), the CloneEZ PCR cloning kit (GeneScript), circular polymerase extension cloning (CPEC) [16,17], polymerase incomplete primer extension (PIPE) [18], quick and clean cloning [19], FastCloning [20], Inverse Fusion PCR cloning [21], and the versatile zero background T-vector system [15]. Among these methods, multifragment DNA cloning can be used for Gateway cloning, In-Fusion cloning, and CPEC.…”

Section: Resultsmentioning

confidence: 99%

Ultra-low Background DNA Cloning System

Nagano¹,

K²

2013

BIO-PROTOCOL

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Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some byproducts. We have developed an ''ultra-low background DNA cloning system'' on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp r ). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp r 59 UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp r 39 UTR. This cassette allowed conversion of the Amp r -containing vector into the yeast/E. coli shuttle vector through use of the Amp r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast-and E. coli-specific ''origins of replication'' to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

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“…coli GlpF was inserted into pBAD/HisA/p15A behind the XhoI site using the polymerase incomplete primer extension (PIPE) cloning method. 17 The primers used to amplify the insert are as follows: forward 5 0 GGA TCC GAG CTC GAG ATG AGT CAA ACA TCA ACC TTG AAA CGC 3 0 and reverse 5 0 CAA AAC AGC CAA GCT TTT ACA GCG AAG CTT TTT GTT CTG AAG G 3 0 . The primers used to amplify the vector are as follows: forward 5 0 CGG CCG ACC TCT GGC ACC TCT GGC ACC 3 0 and reverse 5 0 AAG CTT GGC TGT TTT GGC GGA TGA GAG 3 0 .…”

Section: Cloning Of Controls Used In Cellular Fractionationmentioning

confidence: 99%

“…This construct was made using the PIPE cloning method. 17 The primers used to amplify the vector and which included code for an Nterminal 6ÂHis tag and stop codon are as follows: forward 5 0 CAC CAC CAC CAC CAC CAC TGA AAC AAC AAC AAC AAT AAC AAT 3 0 and reverse 5 0 TCA GTG GTG GTG GTG GTG GTG CGA GCT CGA ATT AGT CTG CGC 3 0 .…”

Section: Cloning Of Controls Used In Cellular Fractionationmentioning

confidence: 99%

“…20 Code for mutD5 was then inserted into pSC101 using the PIPE cloning method. 17 The primers used to amplify the insert are as follows: forward 5 0 AAC CTG TAC TTC CAG CGC CTC CAG CGC GAC AAT AGC GGC CAT C 3 0 and reverse 5 0 GAG TTA ATT AAG TCG CGC CGA CTG AAC TAC CGC TCC GCG TTG TG 3 0 . The primers used to amplify the vector are as follows: forward 5 0 CGC GAC TTA ATT AAC TCC GTC GAA AAC TGC ATG CAT TGC 3 0 and reverse 5 0 CTG GAA GTA CAG GTT CGT TTA ATC ACC AGA ACG GTG GTG 3 0 .…”

Section: Cloning Of Psc101ts/mutd5mentioning

confidence: 99%

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Genetic selection system for improving recombinant membrane protein expression in E. coli

et al. 2009

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A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

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Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Cited by 274 publications

References 28 publications

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Ultra-low Background DNA Cloning System

Genetic selection system for improving recombinant membrane protein expression in E. coli

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