Heterologous expression of Mytilus californianus foot protein three (Mcfp-3) in Kluyveromyces lactis.
Mytilus californianus foot protein three (Mcfp-3) was successfully expressed in the yeast, Kluyveromyces lactis. The first nine amino acids (YPYDVPDYA) from the human-influenza-virus hemagglutinin (HA) protein were fused to the amino terminus of Mcfp-3 (HA-Mcfp-3) to facilitate identification and purification. HA-Mcfp-3 was purified to a concentration of 1mg/L using HA affinity chromatography. The recovered polypeptide was resolved by SDS-PAGE and migrated primarily at 36 kDa, an increase of approximately 29 kDa over the calculated molecular weight of a HA-Mcfp-3 monomer. Significantly, release of Mcfp-3 by enterokinase treatment coincided with the formation of high molecular weight complexes. It is noteworthy that the complexes mimicked the previously reported insolubility of Mcfps found in vivo to denaturing and reducing conditions. These data demonstrate the successful expression of Mcfp-3 in K. lactis and show an intrinsic ability of Mcfp-3 to self-assemble into stable, higher molecular weight forms.
Platko
Joseph D
JD
Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA.
Deeg
Matthew
M
Thompson
Valery
V
Al-Hinai
Zaid
Z
Glick
Hillary
H
Pontius
Kathryn
K
Colussi
Paul
P
Taron
Christopher
C
Kaplan
David L
DL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000494:pharmacology; D006389:Hemagglutinins, Viral / Q000378:metabolism; D006801:Humans; D007251:Influenza, Human; D007716:Kluyveromyces / Q000235:genetics; D008970:Molecular Weight; D049878:Mytilus / Q000033:anatomy & histology* / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016622:Silver Staining; D014760:Viral Fusion Proteins / Q000737:chemistry
D003062:Codon; D006389:Hemagglutinins, Viral; D010446:Peptide Fragments; D011506:Proteins; D014760:Viral Fusion Proteins; D004765:Enteropeptidase