A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins

Kwon, Ki-Ryong; Pieper, Rembert; Shallom, Shamira; Grose, Carissa; Kwon, Erika M.; Do, Yu; Latham, Saeeda; Alami, Hamid; Huang, Shih‐Ting; Gatlin, Christine L.; Papazisi, L.; Fleischmann, Robert; Peterson, Scott N.

doi:10.1016/j.pep.2007.06.006

Cited by 9 publications

(24 citation statements)

References 39 publications

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“…Whole lysate and supernatant fractions were processed on the LabChip GXII (Perkin Elmer, Waltham, MA) to examine expression and solubility level of N-terminal 6xHis-tag fused target proteins. C-terminal CPD-tag fused recombinant proteins were purified using Ni-NTA agarose resin in a 96-well block as previously described [22]. The recombinant proteins were eluted in 200 μL elution buffer (50 mM Tris–HCl, 200 mM imidazole, 300 mM NaCl, 1mM DTT pH 7.8 at 4 °C).…”

Section: Methodsmentioning

confidence: 99%

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

et al. 2017

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BackgroundRecombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols.ResultsIn this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called “the CPD-tag”. The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline.ConclusionpCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0323-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

et al. 2017

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“…We have attempted to strike this ideal by constructing a series of expression vectors that merge the qualities associated with the HaloTag to the ease and efficiency associated with the either LIC or Gateway cloning methods. The Gateway compatible expression vector has the added advantage that it allows investigators to utilize existing entry clone sets which have been produced and made available through public repositories () [14, 18]. We have evaluated the outcomes of a number of protein expression trials using these chimeric vectors.…”

Section: Adaptation Of Halotag To Gateway Expression Vectorsmentioning

confidence: 99%

“…The expression vector, pGW-nHalo, is based on the vector pFN18A which replaced the barnase with the attR recombination cloning sites and ccdB cassette. We also constructed pHis-cHalo another Gateway compatible vector based on pFN20A and T02 (pHis) vectors [14] that contains an N-terminal His-tag and a C-terminal HaloTag. We also constructed a ligation independent cloning vector with a C-terminal HaloTag (pLIC-Halo) based on the pMCSG7 vector backbone [35] and consists of an N-terminal His-tag and a C-terminal HaloTag.…”

Section: Adaptation Of Halotag To Gateway Expression Vectorsmentioning

confidence: 99%

“…Our ability to determine the function of genes places strong demands on a variety of disciplines related to recombinant protein technologies. The large-scale characterization of protein function requires very efficient recombinant proteins production in a high-throughput environment and the necessary automation to perform high-throughput functional screens [13, 14]. Likewise, complementary technologies that broaden the use of recombinant proteins such as labeling methods, sub-cellular localization determination, enzymatic activity and substrate specificity will also need to be developed and advanced if we are to make significant progress.…”

Section: Introductionmentioning

confidence: 99%

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The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Peterson

Kwon²

2013

CCG

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Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

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“…The development of DNA microarray technology and the complete annotation of the S. pneumoniae genome (Dopazo et al, 2001;Hoskins et al, 2001;Tettelin et al, 2001;Lanie et al, 2007), have provided optimal tools to begin to understand the mechanism of pneumoccocal disease at the gene expression (Mascher et al, 2003;McDaniel et al, 2004;Martin-Galiano et al, 2005;Pandya et al, 2005;Marrer et al, 2006;Hendriksen et al, 2007;McKessar and Hakenbeck, 2007) and proteomic levels Lee et al, 2006;Kwon et al, 2007). Here we report the global transcriptional profiling of S. pneumoniae ATCC 6304 serotype 4 after extended serial passage of 50× (50P) and 100× (100P) on sheep blood agar (BA) plates.…”

Section: Introductionmentioning

confidence: 99%

Global transcription profiling and virulence potential of Streptococcus pneumoniae after serial passage

Pandya

Sinha

Luxon

et al. 2009

Gene

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A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins

Cited by 9 publications

References 39 publications

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Global transcription profiling and virulence potential of Streptococcus pneumoniae after serial passage

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