Biomedical subjects

Daniel Christ

Publications and source records attributed to Daniel Christ.

3 recordsLinked to original sources

PubMed2006

Tapping diversity lost in transformations--in vitro amplification of ligation reactions.

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>10(9) transformants in Escherichia coli and 10(5)-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.

Bacillus Phages↗

PubMed2006

Identification of protein domains by shotgun proteolysis.

The identification of protein domains within multi-domain proteins is a persistent problem. Here, we describe an experimental method (shotgun proteolysis) based on random DNA fragmentation and protease selection of the encoded polypeptides on phage for this purpose. We applied the method to the Escherichia coli genome and identified 124 protease-resistant fragments; several were re-cloned for expression as soluble fragments in bacteria, and corresponded to autonomously folding units with folding energies similar to natural protein domains (DeltaG(u)=3.8-6.6 kcal/mol). Structural information was available for approximately half of the selected proteins, which corresponded to compact, globular and domain-sized units that had been derived from a wide range of protein superfamilies. Furthermore, boundaries of the selected fragments correlated with domain boundaries as defined by bioinformatics predictions (R2=0.82; p=0.016). However, predictions were incomplete or entirely lacking for the remaining fragments, reflecting the limited proteome coverage of current bioinformatics methods. Shotgun proteolysis therefore provides a means to identify domains and other autonomously folding units on a genome-wide scale, without any prior knowledge of sequence or structure. Shotgun proteolysis should be particularly valuable for structural studies of proteins and represents a high-throughput alternative to the classical limited proteolysis method for the isolation of stable components of multi-domain proteins.

Escherichia coli↗

PubMed2003

Identification of functional similarities between proteins using directed evolution.

Protein sequences are often highly redundant and evolution can change them beyond recognition. It can therefore be difficult to identify proteins with functional or structural similarities by inspection of their sequences. Here we have used an experimental evolutionary approach to detect hidden similarities between the antisense RNA-binding protein Rop and other proteins. We created an envelope of functional Rop mutants by combinatorial mutagenesis, used the compilation of mutant sequences to search a database of protein structures, and thereby identified a segment of the enzyme valyl-tRNA-synthetase (ValRS). Further inspection revealed that the structures of the RNA-binding sites of both proteins are highly related, as indeed are the RNA ligands. From the known 3D structure of the ValRS in complex with tRNA, we were able to build a model of an RNA hairpin pair in complex with Rop that has proved to be consistent with the biochemical and NMR data for the interaction between Rop and RNA hairpins. We suggest that this approach (mutational envelope scanning), by generating sequence information de novo, can help uncover hidden similarities between proteins.

Amino Acids↗