Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF Mass Spectrometry.
An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZalphaA vector under the control of AOX1 promoter, which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native N- and C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.
Calik
Pinar
P
Chemical Engineering Department, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531 Ankara, Turkey. pcalik@metu.edu.tr
Orman
Mehmet Ali
MA
Celik
Eda
E
Halloran
S Mitchell
SM
Calik
Güzide
G
Ozdamar
Tunçer H
TH
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone