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1.
J Struct Biol ; 177(2): 329-34, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22245778

RESUMO

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.


Assuntos
MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , Engenharia de Proteínas , Clonagem Molecular , Cristalização , Cristalografia , Escherichia coli , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
2.
Protein Sci ; 15(10): 2356-65, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17008718

RESUMO

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.


Assuntos
Técnicas de Química Combinatória , Estrutura Terciária de Proteína , Proteínas/química , Biblioteca Gênica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Solubilidade
3.
Angew Chem Int Ed Engl ; 40(21): 3948-3959, 2001 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-12404465

RESUMO

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.

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