Molecular cloning of the thymus-specific parvalbumin known as avian thymic hormone: isolation of a full length cDNA and expression of the recombinant protein in Escherichia coli.
The complete coding sequence of the thymus-specific parvalbumin called avian thymic hormone (ATH) has been cloned into Escherichia coli. The translated amino acid sequence was found to be identical to the sequence of map turtle parvalbumin at 90 of 108 positions. Northern blot analysis of thymic RNA indicated a transcript length of approximately 1050 bp. However, the ATH cDNA probe failed to hybridize to poly(A)+ RNA from chicken leg muscle, a further indication that avian thymic hormone is distinct from the muscle-associated parvalbumin previously isolated from chicken. Southern analysis of chicken genomic DNA suggests the presence of a single copy of the ATH gene, and the absence of hybridization between an ATH cDNA fragment and genomic DNA from rat and rabbit is confirmatory evidence that ATH expression is restricted to avian species. One of the full length ATH cDNA clones harbored an insert that lacked all 5' noncoding sequences. This cDNA was inserted without further alteration into the prokaryotic expression vector, pKK223-3. The resulting construction, which contains eleven base pairs between the Shine-Dalgarno sequence and the initiation codon, affords reasonably high levels of expression in E. coli. In most respects, recombinant ATH mimics the tissue-derived protein, retaining a similarly high affinity for Ca2+ ion (KCa = 14 +/- 5 nM). However, in contrast to ATH isolated from chicken thymus tissue, the N-terminal alanine of recombinant ATH is unacetylated. As a result, the isoelectric point is shifted upward from 4.3 to approximately 4.8.
Palmisano
W A
WA
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Henzl
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010320:Parvalbumins / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics*; D015183:Restriction Mapping
D010320:Parvalbumins; D011994:Recombinant Proteins; C070978:avian thymic hormone; D004247:DNA