Production and Characterization of Recombinant Human Lactoferrin in Transgenic Javanica Rice

Abstract

The objective of the present study was to develop a functional crop that could contribute to the maintenance and improvement of human health by the introduction of the human lactoferrin (hLF) gene. Lactoferrin is an 80-kDa iron-binding glycoprotein that has been considered to play many biological roles, including the regulation of iron absorption, protection against microbial and virus infection, stimulation of the immune system and cellular growth promotion. We introduced two different constructs containing either the native signal peptide from human lactoferrin (pIG211) or the signal peptide from rice glutelin (pIG200) fused to mature human lactoferrin into Javanica rice cv. Rojolele by using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the maize ubiquitin-1 promoter was detected in all the tissues of the transgenic plants. We found that the transgenic rice plants IG200R produced a considerable amount of recombinant hLF (rhLF) in seeds, accounting for approximately 15% of the total soluble protein (TSP). RhLF was purified from mature seeds by cation-exchange chromatography. Amino acid sequencing confirmed that the N-terminal sequences of rhLF for both constructs were identical with those of native LF from human milk. Rice rhLF showed a slightly smaller molecular weight than the native hLF. Immunofluorescence microscopy revealed that rhLF was located in the intracellular and intercellular regions of endosperm cells. Protein extracts from transgenic rice seeds exhibited an antibacterial activity against Bacillus subtilis ATCC6633. The use of signal peptides and a constitutive ubiquitin-1 promoter for successful production of transgenic Javanica rice expressing a high level of rhLF was examined.

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…The demand for LF has stimulated the development of various recombinant technologies for an efficient and economical production of LF. One successful product is the recombinant rice lactoferrin (rLF; Nandi et al 2005;Rachmawati et al 2005). The rLF has the same amino acid sequence and three-dimensional structure as hLF.…”

High-level expression and production of human lactoferrin in Pichia pastoris

“…To meet these aspects, large amounts of hLF are required. Although hLF has been expressed in various systems [1,10,12,13,16,19,26,28], the potential nutritional and therapeutic application of hLF has not been exploited so far due to the lack of an inexpensive method for the production of sufficient quantities of functional LF for commercial use. Ying et al [28] have reported the production of rhLF in the methylotrophic yeast Pichia pastoris, but the level was moderate, with the highest production of 115 mg·L −1 .…”

Genetic and Molecular Analysis of Transgenic Rice cv. Rojolele Expressing Lactoferrin

“…Transgenic rice cv. Rojolele expressing recombinant human lactoferrin gene was obtained through Agrobacterium-mediated transformation (Rachmawati et al, 2005). Transgenic lines TR-7, TR-8, and TR-10 carried mature human lactoferrin gene was fused to the DNA sequence encoding the rice glutelin signal peptide with the constitutive maize ubiquitin-1 promoter.…”

“…Rojolele has become a popular local rice variety in Central Java and is distributed throughout Indonesia because it has an aromatic fragrance and a delicious taste (Mudjisihono et al, 2002). Rachmawati et al (2005) have been introduced the lactoferrin gene into Javanica rice cv. Rojolele.…”

“…The expression levels of rhLF significantly increased in the mature seeds. A considerable amount of recombinant hLF (rhLF) in seeds were approximately 15% of the total soluble protein (Rachmawati et al, 2005). However, protein expression level depends on both production and stability.…”