Degree Type

Dissertation

Date of Award

2012

Degree Name

Doctor of Philosophy

Department

Agronomy

First Advisor

Kan Wang

Abstract

Maize is not only one of the world's most important crops, but it is also the first field-grown plant-based recombinant expression system developed for commercial purposes. With advantages of low cost, high yield, high protein stability, and well-developed transformation technology over other plant systems, maize is considered to be an ideal recombinant production system. However, there are three major challenges limiting the use of maize for producing recombinant proteins: 1) maize lacks mammalian-like post-translational modification ability that may be required for pharmaceutical and industrial proteins; 2) transgenic pollen containment in open-field production; and 3) low expression of recombinant products. The aim of the present study was to develop strategies to safely produce recombinant proteins with high yield and adequate modification in maize seeds.

Firstly, we showed that the recombinant human collagen type I alpha 1 (rCIα1) with high ratio of hydroxylated prolines (Hyp) similar to native human collagen was expressed in maize seeds co-expressing a recombinant human prolyl 4-hydroxylase (rP4H). The hydroxylated rCIα1 had a markedly enhanced thermal stability compared to non-hydroxylated rCIα1.

Secondly, we demonstrated that a cytoplasmic male-sterile maize system could be used for open-field production of maize-derived recombinant protein to minimize contamination risk caused by pollen drift. Two strategies were presented: the transgene was directly transformed into a male-sterile line via biolistic- or Agrobacterium -mediated methods, or introgressed from male-fertile to male-sterile germplasm by conventional breeding. The male-sterile transgenic maize seed from the second strategy can be used for open-field production by using a non-transgenic maize pollen donor to produce 100% transgenic seeds after six seasons of breeding.

Thirdly, we examined the feasibility of producing a small peptide in maize seeds. A designed 22 amino acid peptide biosurfactant GAM1 was fused to the C-terminus of a fluoresce protein, the red fluorescent protein (RFP), or a maize seed storage protein, the 22-kDa α-zein, to avoid proteolytic degradation in the cell. While red fluorescence could be detected in seeds carrying RFP fusion construct, we failed to detect the presence of GAM1 despite using various protein concentration and detection methods. Strategies for producing this peptide in maize will need to be redesigned.

Lastly, we evaluated two strategies for enhancing recombinant protein expression in maize seeds. In the first strategy, we generated a line with reduced seed storage protein γ-zein by RNA interference (γRNAi). Then we crossed this line with a transgenic line expressing green fluorescent protein (γGFP). The GFP levels in subsequent generations were decreased in the γGFP/γRNAi seeds, which was opposite to what we expected. In the second strategy, we generated a line (oxPBF) constitutively overexpressing the transcription factor prolamin-box binding factor (PBF) that plays regulatory roles to the 27-kDa γ-zein promoter. We intend to cross the oxPBF line with transgenic lines expressing the GFP or recombinant proteins driven by the 27-kDa γ-zein promoter. It is hoped that the PBF overexpression may enhance the production of foreign protein under the γ-zein promoter control.

This work indicated that maize has the potential to produce recombinant proteins with mammalian-like post-translational modifications and can be used for open-field production when using a cytoplasmic male-sterile breeding system.

Copyright Owner

Xing Xu

Language

en

File Format

application/pdf

File Size

202 p.