RESUMO
We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Biblioteca de Peptídeos , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Bacteriófago M13/fisiologia , Sequência de Bases , Capsídeo/genética , Capsídeo/metabolismo , Ensaio de Imunoadsorção Enzimática , Variação Genética/genética , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Soros Imunes/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas/genética , Receptores da Somatotropina/química , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estreptavidina/química , Estreptavidina/genética , Estreptavidina/imunologia , TermodinâmicaRESUMO
BACKGROUND: Full-thickness macular holes (FTMH) are an important cause of visual loss in older patients. Recognition of tangential vitreofoveal traction as the mechanism of hole formation has led to the use of vitrectomy and gas tamponade as a treatment for macular holes. The use of growth factors as an adjunctive therapy to promote chorioretinal adhesion and hole closure has also been advocated. METHODS: Forty-four consecutive patients undergoing vitrectomy, posterior hyaloid removal, gas injection and installation of autologous serum for FTMH were studied prospectively. Patients' age, sex, duration of symptoms, best corrected pre- and post-operative Snellen visual acuities, best corrected acuity in the fellow eye, stage of macular hole, post-operative closure or persistence of the macular hole, and intra- and post-operative complications were recorded. RESULTS: Of 43 patients with adequate follow-up (minimum 6 weeks, mean 6.1 months) anatomical closure of the macular hole was achieved in 29 eyes (67%). The average visual improvement in these eyes was 2.7 lines. Twenty eyes (69%) with closed holes had visual acuity of 6/12 or better (47% of all patients undergoing surgery). Of the 14 eyes (33%) that failed, the average loss of vision was 0.4 lines and all these eyes had vision of 6/36 or worse. CONCLUSIONS: In this series, surgical treatment of FTMH with vitrectomy, gas injection and autologous serum resulted in closure of the macular hole in two-thirds of patients and restored central vision in about one-half of patients. These results compare favourably with previously reported results of macular hole surgery. The answer to the question whether the use of autologous serum leads to a higher closure rate than surgery with gas tamponade alone requires information from randomised treatment trials, one of which is currently in progress.