Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm.
Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.
Farinas
Cristiane S
CS
Laboratório de Engenharia de Bioprocessos, Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, CP 6066, CEP 13083-970, Campinas SP, Brazil.
Leite
Adilson
A
Miranda
Everson A
EA
D005591:Chemical Fractionation / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D006801:Humans; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011384:Proinsulin / Q000096:biosynthesis* / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods; D012639:Seeds / Q000378:metabolism*; D012996:Solutions; D014462:Ultrafiltration / Q000379:methods; D014867:Water / Q000737:chemistry*; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D012996:Solutions; D014867:Water; D011384:Proinsulin