In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Directed evolution of novel binding proteins

Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.

Molecular and Biotechnological Aspects of Microbial Proteases

A progressive alignment method is described that utilizes the Needleman and Wunsch pairwise alignment algorithm iteratively to achieve the multiple alignment of a set of protein sequences and to construct an evolutionary tree depicting their relationship. The sequences are assumed a priori to share a common ancestor, and the trees are constructed from difference matrices derived directly from the multiple alignment. The thrust of the method involves putting more trust in the comparison of recently diverged sequences than in those evolved in the distant past. In particular, this rule is followed: “once a gap, always a gap”. The method has been applied to three sets of protein sequences: 7 superoxide dismutases, 11 globins, and 9 tyrosine kinase-like sequences. Multiple alignments and phylogenetic trees for these sets of sequences were determined and compared with trees derived by conventional pairwise treatments. In several instances, the progressive method led to trees that appeared to be more in line with biological expectations than were trees obtained by more commonly used methods.

Progressive sequence alignment as a prerequisite to correct phylogenetic trees.

Glucosinolates are sulfur-rich, anionic natural products that upon hydrolysis by endogenous thioglucosidases called myrosinases produce several different products (e.g., isothiocyanates, thiocyanates, and nitriles). The hydrolysis products have many different biological activities, e.g., as defense compounds and attractants. For humans these compounds function as cancer-preventing agents, biopesticides, and flavor compounds. Since the completion of the Arabidopsis genome, glucosinolate research has made significant progress, resulting in near-complete elucidation of the core biosynthetic pathway, identification of the first regulators of the pathway, metabolic engineering of specific glucosinolate profiles to study function, as well as identification of evolutionary links to related pathways. Although much has been learned in recent years, much more awaits discovery before we fully understand how and why plants synthesize glucosinolates. This may enable us to more fully exploit the potential of these compounds in agriculture and medicine.

Biology and biochemistry of glucosinolates

For several decades researchers have been studying the biosynthesis and regulation in plants of specific chemicals associated with defense against pests and pathogens. For years many of these chemicals were first considered to be "secondary plant compounds" with unknown functions. Many, if not most, of these "secondary compounds" are now considered to be defensive chemicals that are acting either alone or in concert to contribute to the resistances of plants against insects or pathogens (86,87, 118, 144, 150). These defensive chemicals are found as either constitutive components in various plant tissues (118, 144) or are synthesized in response to attacking pests or pathogens (7, 32,39,45,50,60,63,64,66, 74, 120, 144, 151, 178, 182). The inducible chemicals, which can also occur constitutively, include such complex sub­ stances as antibiotics, alkaloids, and terpenes, as well as proteins such as enzymes, enzyme inhibitors, and lectins. These chemicals have provided interesting systems for studying inducible plant defense. In various plants, they can be newly synthesized in response to pest and/or pathogen attacks, which generate interand intracellular signals to activate genes that code for their production. Many of these systems, therefore, are being studied to help further our fundamental knowledge about the biochemistry of signal genera-

Protease Inhibitors in Plants: Genes for Improving Defenses Against Insects and Pathogens

Biochemical and molecular characterization of three barley seed proteins with antifungal properties.

Biochemical and molecular characterization of a barley seed beta-glucosidase.

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes.

A solid-phase assay for the complete subsite mapping of the active site of endoproteases has been developed. A library of resin-bound protease substrates was synthesized both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer type of resin that allows proteases to diffuse into the interior and perform their catalytic activity. Anthranilic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet mixing of the beads and parallel washing procedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the preferred sequences, their cleavage bond, and a semiquantitative estimation of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.

https://www.pnas.org/content/pnas/91/8/3314.full.pdf

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

A protein inhibitor of endogenous α-amylase 2 has been isolated from germinated barley by glycogen precipitation followed by cation-exchange chromatography. Preliminary kinetic analysis showed a mixed type mechanism of inhibition with an apparent Ki of 4×10−8M. The inhibitor formed well-defined complexes with barley malt α-amylase 2 and co-purified with the α-amylase by cycloheptaamylose affinity chromatography of glycogen precipitates. The inhibitor was inactive towards α-amylases from sorghum malt, hog pancreas, Aspergillus oryzae, and Bacillus subtilis. The amino acid composition and molecular weight near 21,000 were found to be the same as those of both “band-2 protein” previously identified in preparations of barley malt α-amylase and a specific subtilisin inhibitor from barley. Inhibition experiments confirmed that the malt α-amylase inhibitor is a strong inhibitor of subtilisin Carlsberg. Measurements of α-amylase activity in the presence of equimolar amounts of inhibitor and subtilisin showed that the inhibitor is “double headed”. The inhibitory activity towards α-amylase was lost after treatment of the inhibitor at 70 °C for 15 min. Isoelectric focusing patterns confirmed that the partially heat-labile, basic α-amylase isozymes (pI=6.6) of barley malt are complexes of α-amylase 2 (pI=6.2) and the inhibitor (pI=7.2). Evidence is presented to suggest that proteins with properties similar to those of the barley inhibitor are present in other cereals including wheat and rye. Possible in vivo functions and some practical aspects of the barley inhibitor are discussed.

/pdf/barley-a-amylase-subtilisin-inhibitor-i-isolation-and-4xlwdhmtmb.pdf

Ib Svendsen

Papers

Biochemical and molecular characterization of three barley seed proteins with antifungal properties.

Biochemical and molecular characterization of a barley seed beta-glucosidase.

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes.

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization