RESUMO
The use of tobacco plants as a novel expression system for the production of human homotrimeric collagen I is presented in this report. Constructs were engineered from cDNA encoding the human proalpha1(I) chain to generate transgenic tobacco plants expressing collagen I. The recombinant proalpha1(I) chains were expressed as disulfide-bonded trimers and were shown to fold into a stable homotrimeric triple helix. Moreover, the recombinant procollagen was subsequently processed to collagen as it occurs in animals. Large amounts of recombinant collagen were purified from field grown plant material. The data suggest that plants are a valuable alternative for the recombinant production of collagen for various medical and scientific purposes.
Assuntos
Nicotiana/genética , Plantas Tóxicas , Pró-Colágeno/genética , Sequência de Aminoácidos , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Pró-Colágeno/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , TripsinaRESUMO
Production and characterization of human lactoferrin (hLf) in transgenic tobacco is reported. We have engineered two constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin encoding cDNA. N-terminal sequences of rhLf purified from tobacco were identical to Lf from human milk for both constructs. The tobacco rhLf presents a molecular mass closely identical to native protein. Overall sugar composition shows the presence of plant specific xylose while sialic acid is absent. Binding parameters of the recombinant molecule to both Jurkat lymphoblastic T-cells or HT29-18-C1 enterocytes are similar to those of human lactoferrin isolated from milk.