Production of functional human α 1 -antitrypsin by plant cell culture

Abstract

Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

“…Cleavage of the RCL at 354 E has been found to be typical of the lysosomal Cys proteinase cathepsin L (Johnson et al, 1986) and nontarget Ser proteases (Nelson et al, 1998). Considerable amounts of truncated protein were also obtained in previous attempts to express recombinant A1AT in rice (Oryza sativa) cells (Terashima et al, 1999;Huang et al, 2001;Trexler et al, 2002;McDonald et al, 2005), N. benthamiana (Sudarshana et al, 2006), and Nicotiana tabacum chloroplasts (Nadai et al, 2009). Nevertheless, A1AT produced in transgenic tomato (Solanum lycopersicum) plants does not show degradation (Jha et al, 2012).…”

Plant Bioreactors for Pharmaceuticals

“…Plant suspension cultured cells provide a fast system for producing secondary metabolites, biologically active recombinant proteins and antibodies (Francisco et al, 1997;Terashima et al, 1999;Torres et al, 1999). The recombinant proteins expressed can either be transported to subcellular organelles or be secreted into extracellular space via the default pathway in plant suspension cultured cells (Denecke et al, 1990;Liu et al, 1997).…”

Bioreactor Engineering For Recombinant Protein Production Using Plant Cell Suspension Culture

“…For rice cell culture, Trexler et al [16] reported doubling time of 1.5 ~ 1.7 days for a transgenic rice cell culture expressing human 1 -antitrypsin. Terashima et al [15], on the other hand, reported a very long doubling time of 6-7 days in their transgenic rice cell cultures expressing human 1 -antitrypsin. Maximum specific oxygen uptake rate was 0.78 ~ 0.84 mmol O 2 /(gdw h) in the transgenic rice cell culture reported by Trexler et al [16]; 0.4 ~ 0.5 mmol O 2 /(gdw h) for the transgenic tobacco NT-1 cells expressing GUS [53].…”

“…A number of inducible promoters have been used for expressing recombinant proteins in plant suspension cultures. The rice -amylase (RAmy3D) promoter which is induced by sugar starvation was used in rice cell cultures to express recombinant 1 -antitrypsin [15,16] and recombinant hGM-CSF [5]; the Arabidopsis thaliana heat-shock (HSP18.2) promoter [60], the tomato light inducible rbcS promoter [61], the methyl jasmonate inducible potato cathepsin D inhibitor (CDI) promoter [59], the glucocorticoid-inducible GVG promoter [62], the sweet potato oxidative stress-inducible peroxidase (POD) promoter [63], and the abscisic acid, tetracycline, and copper inducible promoters [64], have all been examined in tobacco cell cultures for recombinant protein production. In order to optimize the efficiency of an inducible gene expression system, it is necessary to examine the inducer concentration and timing of inducer addition.…”

“…Some notable examples are the expression of a secretory anti-phytochrome single-chain Fv (scFv) antibody [6], a TMV-specific recombinant full-size antibody [7], a mouse IgG1 recognizing a cell-surface protein of Streptococcus mutants [8], and a mouse scFv [7,9], all using tobacco suspension culture. A number of therapeutic proteins have also been expressed in plant cell cultures, including Hepatitis B surface antigen (HBsAg) [10], human cytokines such as Interleukin IL-2, IL-4 [11], IL-12 [12], and GM-CSF [5,13], ribosome-inactivating protein [14], and human 1 -antitrypsin [15,16]. Readers are also referred to other comprehensive reviews on the subject of recombinant protein expression in plant tissue cultures [2,4,17].…”