Human growth hormone variants

G. Teshima and E. Canovadavis, Separation of oxidized human growth hormone variants by reversed-phase HPLC-effect of mobile phase pH and organic modifier, J. Chromatogr., 625 201 (1992). 

Fig. 1. Primary stmcmre of the human growth hormone family of proteins. GH-N = pituitary GH GH-V = placental GH variant ...

Lastly, it has been shown that in rare cases trisulfide bridges (Cys-SSS-Cys) can be found as hydrophobic variants of recombinant proteins. The experimental mass of such a trisulfide-bridged peptide would be increased by 32 amu. Andersson et al.125 have described this phenomenon for recombinant human growth hormone during expression in E. coli. However, the receptor binding properties were not affected by this trisulfide modification. 

Andersson C., Edlund P.O., Gellerfors P, Hansson Y., Holmberg E., Hult C., Johansson S., Kordel J., Lundin R., Mendel-Hartvig I., Noren B., Wehler T., Wid-malm G., and Ohman J. (1996), Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli, Int. J. Peptide Protein Res. 47(4), 311-321. 

Proteolytic secretion variants of recombinant human growth hormone were isolated by anion-exchange HPLC but compared analytically to the reference standard using reversed-phase (C4) HPLC with an ammonium bicarbonate/acetonitrile mobile phase rather than the more commonly used TFA/acetonitrile mobile phase [297]. 

The idea of using CIEF to separate bound from free antibody was realized for the first time by Shimura et al. [25], These authors quantified human growth hormone (hGH) using tetramethylrodamine/iodoacetamide-labeled anti-hGH Fab fragment. Excess fluorescendy labeled Fab was added to solutions with variable amounts of hGH and, upon incubation, Fab -Ag complex was separated from the excess Fab by CIEF. LIF detection was used, which provided a very low detection limit of 5 x 10 12 M of methionyl hGH. In this report different forms of Ag were detected simultaneously in one run, since complexes of labeled Fab with non-, mono-, and di-deaminated variants of met-hGH exhibited different isoelectric points and hence could be resolved by CIEF. 

Gunningham, B. C., and Wells, J. A. (1991). Rational design of receptor-specific variants of human growth hormone. Proc. Natl. Acad. Sci. USA 88, 3407-3411. 

Pal, G., Kossiakoff, A. A., and Sidhu, S. S. (2003). The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alaninescanning mutagenesis Insights into the mechanisms responsible for improved affinity./ Mol. Biol. 332(1), 195-204. 

Schiffer, C., Ultsch, M., Walsh, S., Somers, W., De Vos, A. M., and Kossiakoff, A. (2002). Structme of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor Evidence for strong structural coupling between receptor binding sites./. Mol. Biol. 316(2), 277-289. 

As an illustration of HIC technique, the recombinant human growth hormone (hGH) and methionyl hGH (met-hGH) were well-separated by the HIC technique [14]. The optimized conditions were found to be IM ammonium phosphate dibasic, pH 8.0/propanol (99.5 0.5) and 0.1 M sodium phosphate dibasic, pH 8.0/propanol (97.5 2.5) for mobile phase A and B, respectively, with a descending gradient from 100% A to 100% B in 30 minutes at a column (TSK-phenyl 5PW, 75 x 7.5 mm) temperature of 30°C. Note that the addition of a small amount of propanol as organic modifiers significantly decreases elution time while maintaining resolution and efficiency. This HIC method allowed separation of several hGH variants from the main hGH peak while retaining their native structures. 

The classical parameters of >90% talc adsorption and TCA precipitation, with 3% agreement between the talc and TCA values, and <25% binding to resin are appropriate for polypeptide hormones with a certain chemical composition, including most anterior pituitary hormones. We have found, so far, two exceptions to the above parameters the S variant form of human growth hormone (hGH-S) and human FSH (all preparations). These are acidic proteins and show a decreased adsorption to talc and an increased binding to resin. 

GH assays now use mouse monoclonal antibodies, and some of these assays are able to discriminate GH variants. Most procedures use recombinant-derived GH for labeling with a tracer and calibration material. The latter is usually prepared gravimetricaUy and verified by comparison with an international reference preparation (IRP), such as the World Health Organization s (WHO s) international standard, IRP 80/505 human growth hormone recombinant (hGHr), which has a potency of 3.3IU/mg of r-hGH, or other standard preparations, such as WHO IRP 66/217 or 88/624. The assay diluent, however, can vary considerably from one assay to another and is a potential source of bias some commercial kits use phosphate-buffered saline, and others use horse, calf, or human serum. 

R17. Rudman, D., Kutner, M. H., Blackston, R. D., Cushman, R. A., Bain, R. P., and Patterson, J. H., Children with normal-variant short stature Treatment with human growth hormone for six months. N. Engjt. J. Med. 305, 123-131 (1981). 

J.M. Haugh, Mathematical model of human growth hormone (hGHJ-stimulated cell proliferation explains the efficacy of hGH variants as receptor agonists or antagonists, Biotechnol. Prog. 2004, 20,1337-1344. 

Baumann G, Shaw M A (1990). Plasma transport of the 20,000 dalton variant of human growth hormone (20K) Evidence for a 20K-specific binding site. J. Clin. Endocrinal. Metab. 71 1339-1343. 

Oxidized human growth hormone (hGH) variants were preparatively separated on a PLRP-S column using a 100-min 34/66 -> 39/61 n-propyl alcohol/water (25 mM ammonium acetate buffer at pH 7.5) gradient Excellent resolution was obtained for the oxidized and native hGH using a IS mg load [486]. Further identification of the fractions was done by LC/MS. 

Masuda, N., Watahiki, M., Tanaka, M., et al (1988) Molecular cloning of cDNA encoding 20kDa variant human growth hormone and the alternative splicing mechanism. Biochimica et Biophysica Acta, 949,125-131. 

Kohler, M., Thomas, A., Puschel,K.,effl/. (2009) Identification of human pituitary growth hormone variants by mass spectrometry. Journal of Proteome Research, 8,1071-1076. 

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