RESUMO
A fusion protein construct consisting of the short form of human fibroblast growth factor (FGFR) fused to the heavy chain of mouse IgG1 was used to screen four phage display libraries displaying 8, 13, 38 and 43 amino acids at the amino terminus of the bacteriophage M13 gene III minor coat protein. Phage with specific FGFR binding activity were isolated from the 13, 38 and 43 mer libraries. One of the highest affinity phage clones from the 13mer library was chosen to be further evolved by oligonucleotide saturation mutagenesis. We have isolated evolved sequences that have approximately 8 times the relative binding affinity of the parent sequence. The phage clones have a minimum consensus sequence of CR/SXLLXGAPFXXXXC, where X represents positions tolerant of several amino acids. A synthetic peptide based on this sequence specifically inhibits FGF from binding to its receptor in an in vitro ELISA.
Assuntos
Bacteriófagos/genética , Receptores de Fatores de Crescimento de Fibroblastos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Clonagem Molecular , DNA , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imunoglobulina G/genética , Camundongos , Dados de Sequência Molecular , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
Functional complementation of the Saccharomyces cerevisiae glycogen branching enzyme deficiency was screened to isolate human cDNAs that encode this enzyme. Human hepatoma cell line HepG2-derived cDNA libraries using the pAB23BXN yeast expression vector yielded four cDNAs capable of complementing the glc3::TRP1 glycogen branching enzyme mutation. Complementation was recognized by an altered iodine-staining trait. This illustrates that interspecies complementation can be used to isolate rare plasmids from libraries by screening if there is sufficient resolution. The human and yeast glycogen branching enzymes have a 67% identical amino acid sequence over a major portion of their length. The human gene is on chromosome 3.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Saccharomyces cerevisiae/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Cromossomos Humanos Par 3 , Clonagem Molecular , DNA/isolamento & purificação , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The yeast glycogen branching enzyme (EC 2.4.1.18) is shown to be induced in batch culture simultaneously with the onset of intracellular glycogen accumulation. The branching enzyme structural gene (GLC3) has been cloned. Its predicted amino acid sequence is very similar to procaryotic branching enzymes. Northern analysis indicates that GLC3 mRNA abundance increases in late exponential growth phase coincident with glycogen accumulation. Disruption of the branching enzyme structural gene establishes that branching enzyme activity is an absolute requirement for maximal glycogen synthesis.
