Cloning and expression of P60, a conserved surface-localized protein of Mycoplasma hominis, in Escherichia coli.
The clp60 gene encoding P60, a conserved lipoprotein of Mycoplasma hominis, was cloned and sequenced from both the type strain PG21 and the isolate FBG. Both open reading frames were identical in length, comprising 1746 nucleotides. The deduced amino acid sequences differed in 16 out of 582 amino acids. As expected, none of these divergences mapped within the epitope that was recognized by mAb CG4 in all of the 198 isolates of M. hominis analyzed so far. This conserved epitope was narrowed down to amino acids 454 through 464 within the C terminus of P60. For the expression of the recombinant homolog P60, P60rec, in E. coli the TGA codons of clp60 were substituted for TGG codons prior to cloning of clp60 into the expression plasmid pQE41. The expression of P60rec as a fusion protein with dihydrofolate reductase carrying an N-terminal His-tag enabled the purification of large amounts of P60rec in a soluble form.
Henrich
B
B
Institute for Medical Microbiology and Virology, and Center for Biological and Medical Research, Heinrich-Heine-University, Düsseldorf, Germany.
Berns
G
G
Weinhold
M
M
Kitzerow
A
A
Schaal
H
H
Hadding
U
U
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics*; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D019535:Mycoplasma hominis / Q000235:genetics*; D010957:Plasmids; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; C116063:P60 protein, bacteria