Expression of functional recombinant human lysozyme in transgenic rice cell culture.
Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E. coli cells were exposed to recombinant human lysozyme for 60 min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.
Huang
Jianmin
J
Applied Phytologics, Inc., Sacramento, California 95834, USA. jhuang@apinc.com
Wu
Liying
L
Yalda
Dorice
D
Adkins
Yuriko
Y
Kelleher
Shannon L
SL
Crane
Michael
M
Lonnerdal
Bo
B
Rodriguez
Raymond L
RL
Huang
Ning
N
D019470:Biolistics; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D012275:Oryza / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D013696:Temperature; D014162:Transfection
D011994:Recombinant Proteins; D009113:Muramidase