Production of Recombinant Human Placental Variant Growth Hormone in Pichia Pastoris

Abstract

cDNA encoding mature human placental variant growth hormone (HGH-V) was synthesized by retro-transcription polymerase chain reaction (RT-PCR) from total RNA recovered from human term-placenta and cloned in pBluescript plasmid (pBS) in Escherichia coli. cDNA was subcloned into pPIC9, fusing it to the flanking regulatory sequences of the Pichia pastoris alcohol oxidase 1 gene (AOX1) and finally introduced into the genome of this yeast by homologous recombination. The resulting new recombinant strain produced and secreted, towards the culture medium, mature HGH-V, whose activity was demonstrated in cell culture by the Nb2 proliferation assay.

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: Placental growth hormones have no lactogenic activity in humans

“…The assay-dependent outcome was even more pronounced in the three bioassays that measured lactogenic activity. In the most commonly used lactogenic assay, based on rat lymphoma Nb2 cell proliferation, the hGH-N 20K and hGH-V 22K variants were, respectively, 4.5-and 9-fold less potent that hGH-N 22K, as reported by others [31]. In contrast, in Baf/3 cells stably transfected with the long form of rbPRLR, both hGH-Ns were equally potent while the hGH-V 22K was $55-fold less potent.…”

“…The secondary structure of hGH-N 20K and hGH-V 22K was similar to that of hGH-N 22K, indicating proper folding, whereas the a-helix content of hGH-V 20K indicated a slightly different secondary structure. The yields reported here seem to be much higher than those reported previously [4][5][6][7], and substantially higher than the hGH-V 22K preparation in Pichia pastoris reported recently [31].…”

Derivation of a growth hormone gene cassette for goat by mutagenesis of the corresponding bovine construct and its expression in Pichia pastoris

“…Barrera-Saldañ a, unpublished work). As reported in others papers (Palma-Nicolá s et al 2005;Eurwilaichitr et al 2002), the double spacer Glu-Ala-Glu-Al was unnecessary for the release of the mature rchGH from S. cerevisiae a-mating factor signal peptide.…”

“…Five microgram of the SacI-linearized pPIC9-chGH were used to transform Pichia pastoris host strain GS115 (his4) from Invitrogen using the LiCl method previously described (Gietz et al 1996;Palma-Nicolá s et al 2005). Transformation with the SacI-linearized version of pPIC9-chGH favors its insertion into the yeast genome by homologous recombination.…”

“…Primers that annealed at the AOX1 promoter (5¢-GACTGGTTCCAATTGACAAG C-3¢) and transcription terminator (5¢-GCAAA TGGCATTCTGACATTC-3¢), both from Invitrogen, were used. PCR reactions were performed in a MJ Research thermocycler (Watertown, MA) as described by Palma-Nicolá s et al (2005). PCR products were resolved by electrophoresis in 0.7% (w/v) agarose gels and visualized with ultraviolet light after ethidium bromide staining.…”