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1.
Chem Commun (Camb) ; 50(94): 14900-3, 2014 Dec 07.
Article in English | MEDLINE | ID: mdl-25325394

ABSTRACT

Here we demonstrate a methodology, termed protein stapling, for the introduction of covalent constraints into recombinant proteins. Using the azide-alkyne click reaction as the stapling chemistry, we have improved the thermostability of a model leucine zipper protein. Additionally, stapling the core of the small, globular protein G resulted in improved binding to its target, immunoglobulin G.


Subject(s)
Alkynes/chemistry , Azides/chemistry , Bacterial Proteins/chemistry , Amino Acid Sequence , Immunoglobulin G/metabolism , Leucine Zippers , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary
2.
Biotechnol Lett ; 34(1): 91-6, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21956129

ABSTRACT

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Cell Membrane/chemistry , Cellulose 1,4-beta-Cellobiosidase/analysis , Escherichia coli/chemistry , Trichoderma/enzymology , Bacterial Outer Membrane Proteins/genetics , Cell Fractionation , Cellulose 1,4-beta-Cellobiosidase/genetics , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Trichoderma/genetics
4.
J Am Chem Soc ; 131(47): 17078-9, 2009 Dec 02.
Article in English | MEDLINE | ID: mdl-19894713

ABSTRACT

Engineered aminoacyl-tRNA synthetases have been used to enable the incorporation of many unnatural amino acids into recombinant proteins in vivo. In the majority of these studies, the engineered synthetase is harbored on a plasmid while the host retains a wild-type copy of the synthetase in its genome. Herein, we construct a strain carrying a single genomic copy of a methionyl-tRNA synthetase (MetRS) gene, metG*, engineered to enable the incorporation of azidonorleucine (ANL) into proteins. The resulting strain, M15MA metG*, is capable of both supporting robust cell growth and enabling the production of >20 mg/L culture of a recombinant protein, murine dihydrofolate reductase, containing ANL. The extent of replacement of methionine with ANL in this protein is 90%. Using this strain, we also produce ANL-containing OmpC, an outer membrane protein, and demonstrate that the surface of cells displaying this protein can be covalently modified using copper-catalyzed azide-alkyne cycloaddition. Since this mutant MetRS has been introduced into the genome, as opposed to a plasmid, M15MA metG* is genetically stable.


Subject(s)
Escherichia coli/genetics , Genomics , Methionine-tRNA Ligase/genetics , Norleucine/metabolism , Protein Engineering , Methionine-tRNA Ligase/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization
5.
J Mol Biol ; 394(2): 297-305, 2009 Nov 27.
Article in English | MEDLINE | ID: mdl-19766123

ABSTRACT

The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein-protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72-87) peptide and the anti-apoptotic protein Bcl-x(L), the effect of both increasing and decreasing Bak peptide length on Bcl-x(L) binding was investigated. Previous work demonstrated that the Bak(72-87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-x(L). Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-x(L) and Bcl-2 equally well. Approximate binding affinity calculations on these peptide-protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72-87) variants for improved affinity to Bcl-x(L). The best variants obtained from this library exhibit an apparent K(d) to Bcl-x(L) 4-fold lower than that of wild-type Bak(72-87).


Subject(s)
Apoptosis , Cell Membrane/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/metabolism , Animals , Arginine/genetics , Arginine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Flow Cytometry , Humans , Mutagenesis , Peptides/genetics , Peptides/metabolism , Protein Engineering , Tyrosine/genetics , Tyrosine/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-X Protein/genetics
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