Expression of recombinant protein encoded by LOC387715 in Escherichia coli.
LOC387715 is a hypothetical gene located on human chromosome 10q26.13 that is associated with the development of age-related macular degeneration (AMD). The native open reading frame (ORF) of LOC387715 cDNA - LOC387715(ORF), contains a large number of Escherichia coli (E. coli) rare codons (RC) including 5.6% and 15.0% Group-I and IIa translational problem causative (TPC) RCs, respectively, which forms 3 and 4 simple E. coli rare codon clusters (RCC) where RCs are spaced by 1 and 2 respective non-TPC codons and one complex E. coli RCC where RCs and RCCs are spaced by <5 non-TPC codons. We modified the entire 35 E. coli RCs (6, 16 and 13 Group I, IIa and IIb RCs, respectively) present in LOC387715(ORF) with their optimal or sub-optimal synonymous degenerate codons, and the resulted LOC387715(ORF)m was free from Shine-Dalgarno-like sequence (SDLS) and ribosome binding site complementary sequence (RBSCS). SDS-PAGE and Western blotting analysis demonstrated that LOC387715(ORF)m was capable of highly expressing the recombinant protein rLOC387715 in E. coli. Mass spectrometry analysis indicated that the bacterial expressed rLOC387715 contained the correct and expected amino acid (aa) sequence without aa misincorporation, aa missing or frame-shift. The results suggest that high and authentic expression of LOC387715 recombinant protein in E. coli was achieved by the synonymous modification of its native ORF cDNA sequence for all the 3 groups of bacterial RCs and the simultaneous elimination of SDLS and RBSCS sequences.
Chen
Dequan
D
Institute for Retina Research, Presbyterian Hospital, 8210 Walnut Hill Lane, Dallas, TX 75231, USA. chen.dequan@mayo.edu <chen.dequan@mayo.edu>
Langford
Marlyn P
MP
Duggan
Chris
C
Madden
Benjamin J
BJ
Edwards
Albert O
AO
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002879:Chromosomes, Human, Pair 10; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008268:Macular Degeneration; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
C514392:ARMS2 protein, human; D003062:Codon; D011506:Proteins; D011994:Recombinant Proteins