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PubMed1991

New molecular biology methods for protein engineering.

This review outlines recent advances in the application of molecular biological techniques to the study of protein structure and function. The chapter is divided into four main sections: methods for oligonucleotide-directed mutagenesis; mutational strategies for identifying functional residues and domains; systems for expression; and future developments. Few new methods were reported in 1990; however, a number of the papers that appeared represent refinements of previously reported strategies. This review is also published in Current Opinion in Structural Biology 1991, 1:605-610.

Biotechnology↗
PubMed1991

Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.

If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.

Amino Acid Sequence↗
PubMed2003

Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form.

Thromboxane A2 synthase (TXAS) binds to the endoplasmic reticulum membrane and catalyzes both an isomerization of prostaglandin H2 (PGH2) to form thromboxane A2 (TXA2) and a fragmentation of PGH2 to form 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA). TXAS is a non-classic cytochrome P450 in that it does not require molecular oxygen or an external electron donor for catalysis. Difficulty in obtaining crystals from the membrane-bound TXAS prompted us to modify the protein to a soluble form. Results from site-directed mutagenesis, hydropathy analysis, and homology modeling led us to identify a putative membrane association segment near the end of helix F in TXAS. We report here the generation of a soluble form of TXAS by deletion of the amino-terminal membrane-anchoring domain and replacement of the helix F and F-G loop region with the corresponding region of the structurally characterized microsomal P450 2C5. The resultant TXAS/2C5 chimera is expressed in bacteria as a cytosolic and monomeric protein. Addition of an amino-terminal leader sequence to enhance expression and a tetra-histidine segment at the carboxyl-terminus to facilitate purification yielded approximately 4 mg of nearly homogeneous TXAS/2C5 per liter of bacterial culture. The TXAS/2C5 chimera contains heme at nearly a 1:1 molar ratio and catalyzes the formation of TXA2, MDA, and HHT at a 1:1:1 ratio, although with a reduced catalytic activity compared to wild type TXAS. TXAS/2C5 exhibits electronic absorption spectra similar to wild type TXAS and has similar affinities toward distal heme ligands such as imidazole and U44069. The chimera was mono-dispersive and thus is promising for crystallization trials.

Amino Acid Sequence↗
PubMed1997

New protein engineering approaches to multivalent and bispecific antibody fragments.

Multivalency is one of the hallmarks of antibodies, by which enormous gains in functional affinity, and thereby improved performance in vivo and in a variety of in vitro assays are achieved. Improved in vivo targeting and more selective localization are another consequence of multivalency. We summarize recent progress in engineering multivalency from recombinant antibody fragments by using miniantibodies (scFv fragments linked with hinges and oligomerization domains), spontaneous scFv dimers with short linkers (diabodies), or chemically crosslinked antibody fragments. Directly related to this are efforts of bringing different binding sites together to create bispecific antibodies. For this purpose, chemically linked fragments, diabodies, scFv-scFv tandems and bispecific miniantibodies have been investigated. Progress in E. coli expression technology makes the amounts necessary for clinical studies now available for suitably engineered fragments. We foresee therapeutic advances from a modular, systematic approach to optimizing pharmacokinetics, stability and functional affinity, which should prove possible with the new recombinant molecular designs.

Amino Acid Sequence↗
PubMedDate not supplied

Protein engineering.

Explore the source record for details and available documents.

Carboxypeptidases↗
PubMed2006

Protein engineering of formate dehydrogenase.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.

Amino Acid Sequence↗
PubMed2005

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.

Amino Acids↗
PubMed2000

Exploring the possibilities presented by protein engineering.

A combination of classical and powerful new combinatorial genetic techniques allows the redesign of enzyme activities and creation of proteins that are tailored to have specific properties. These technologies have far-reaching consequences for the future design of crop plants and the storage compounds within them.

Directed Molecular Evolution↗
PubMed2001

Human IFN-alpha protein engineering: the amino acid residues at positions 86 and 90 are important for antiproliferative activity.

Human IFN-alpha is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-alpha hybrids (HY) of alpha21a/alpha2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81-95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-alpha. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-alphas.

Amino Acid Sequence↗
PubMed1999

Co-operative effects of protein engineering and vector optimization on high yield expression of functional bivalent miniantibodies in Escherichia coli.

The volumetric yield of functional phosphocholine-binding miniantibodies could be increased in E. coli fermentations by the combination of the following approaches: Firstly, miniantibody mutants with amino acid exchanges in the VH chain leading to improved folding were expressed. Secondly, the expression vector was stabilized by an efficient suicide system to prevent plasmid loss. Thirdly, the cells were grown to high cell densities in a stirred tank reactor.

Amino Acid Substitution↗