Spectroscopic analysis of recombinant human growth hormone in the presence of sucrose and trehalose.

Recombinant human growth hormone (rhGH) is a therapeutic protein, associated with various human diseases, such as growth hormone deficiency. One of the interesting issues in the formulation of therapeutic proteins is excipients like disaccharides. In the current study, we try to compare the effect of sucrose and trehalose on the structure of rhGH in the liquid state at 25°C and 55°C. We use spectroscopic techniques including intrinsic and extrinsic fluorescence, Fourier-transform infrared (FTIR), circular dichroism (CD), dynamic light scattering (DLS), and time-resolved fluorescence. FTIR shows a slight change in the secondary structure of rhGH in presence of the sugars as sucrose is more effective than trehalose. Fluorescence investigations also confirm the enhancements of folding of rhGH and fluorescein isothiocyanate (FITC)-rhGH in presence of sucrose (1.5-fold more than trehalose). Also, we studied sucrose's effect on the rete of aggregation of rhGH using spectroscopy of Congo red, and fluorescence imaging of thioflavin T (ThT)-treated samples. It can be suggested that sucrose facilitates the amyloid formation of rhGH during 20 days of incubation at 37°C. This study will help to understand the growth hormone structural behavior in the liquid state in the presence of sucrose and trehalose in vitro.

Molecular identification, tissue distribution and in vitro functional analysis of growth hormone and its receptors in red-spotted grouper (Epinephelus akaara).

Red-spotted grouper (Epinephelus akaara) is one of the high economic value grouper species, however, the knowledge regarding its growth is limited. In this study, full-length cDNAs of growth hormone (gh) and its receptors (ghr1 and ghr2) were cloned from the pituitary and liver of red-spotted grouper, respectively. Tissue distribution analysis showed that gh mRNA was predominantly expressed in the pituitary. ghr1 mRNA was highly expressed in the liver, muscle, fat and gonad, while ghr2 mRNA expression was ubiquitously high in the peripheral tissues. However, the mRNA expression of both ghr isoforms was relatively low in the central nervous system. Secretory recombinant grouper GH (rgGH) was expressed in yeast Pichia pastoris and verified. HEK293T cells transiently transfected with the GHR isoforms were used to elucidate the receptor-mediated signaling pathways related to growth regulation. rgGH activated rapid phosphorylation of Janus kinase 2, signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated protein kinase 1/2 through GHR1, but only STAT5 was phosphorylated via GHR2. rgGH strongly activated STAT5 phosphorylation and significantly stimulated ghr1, ghr2 and insulin-like growth factor (igf1, igf2) mRNA expression in primary cultured hepatocytes. Data showed that the recombinant protein rgGH played effects on igf1/2 mRNA expression via GHR-mediated signaling pathways. Our findings provide essential information about GH and GHRs characteristics in red-spotted grouper.

Disulphide-mediated site-directed modification of proteins.

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.

Identification of Binding Sites on Human Serum Albumin for Somapacitan, a Long-Acting Growth Hormone Derivative.

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.

GH and GHR gene cloning, expression and their associations with growth-related traits of the barbel chub (Squaliobarbus curriculus).

Growth hormone (ScGH) and growth hormone receptor (ScGHR) genes from the barbel chub (Squaliobarbus curriculus), in addition to their cDNAs, were cloned. The associations between their mRNA expression levels and growth-related traits were analysed, and the differences in the levels of expression of growth regulation-related genes between the largest and smallest individuals were compared. The full-length 1182-bp cDNA of ScGH contained a 633-bp open reading frame (ORF), and the length of the gene had 2492 bp. The full-length 2825-bp cDNA of ScGHRa contained a 1818-bp ORF, and the gene had 6970 bp. The full-length 2822-bp cDNA of ScGHRb contained a 1737-bp ORF, and the gene had 8149 bp. Quantitative real-time PCR revealed that ScGH was only expressed in the pituitary. ScGHRa was expressed predominantly in muscle, and the expression level of ScGHRb was the highest in the liver. The ScGHRa mRNA levels in the muscle were significantly negatively correlated with the caudal peduncle length. However, no correlation between growth-related traits and ScGH and ScGHRb expression levels were found. Pituitary ScGH, liver GHRb and liver insulin-like growth factor I (igf-1) expression levels were significantly higher in the largest individuals than those in the smallest S. curriculus individuals. Contrarily, the largest individuals had significantly lower expression levels of muscle igf-1 and liver myog than the smallest individuals. Overall, our results provide novel molecular information for growth-regulation study of S. curriculus.

Nickel Sensitivity Is Associated with GH-IGF1 Axis Impairment and Pituitary Abnormalities on MRI in Overweight and Obese Subjects.

Nickel (Ni) is a ubiquitous metal, the exposure of which is implied in the development of contact dermatitis (nickel allergic contact dermatitis (Ni-ACD)) and Systemic Ni Allergy Syndrome (SNAS), very common among overweight/obese patients. Preclinical studies have linked Ni exposure to abnormal production/release of Growth Hormone (GH), and we previously found an association between Ni-ACD/SNAS and GH-Insulin-like growth factor 1 (IGF1) axis dysregulation in obese individuals, altogether suggesting a role for this metal as a pituitary disruptor. We herein aimed to directly evaluate the pituitary gland in overweight/obese patients with signs/symptoms suggestive of Ni allergy, exploring the link with GH secretion; 859 subjects with overweight/obesity and suspected of Ni allergy underwent Ni patch tests. Among these, 106 were also suspected of GH deficiency (GHD) and underwent dynamic testing as well as magnetic resonance imaging for routine follow up of benign diseases or following GHD diagnosis. We report that subjects with Ni allergies show a greater GH-IGF1 axis impairment, a higher prevalence of Empty Sella (ES), a reduced pituitary volume and a higher normalized T2 pituitary intensity compared to nonallergic ones. We hypothesize that Ni may be detrimental to the pituitary gland, through increased inflammation, thus contributing to GH-IGF1 axis dysregulation.

Effect of Iron Oxide Nanoparticles on the Oxidation and Secondary Structure of Growth Hormone.

Oxidation of therapeutic proteins (TPs) can lead to changes in their pharmacokinetics, biological activity and immunogenicity. Metal impurities such as iron are known to increase oxidation of TPs, but nanoparticulate metals have unique physical and chemical properties compared to the bulk material or free metal ions. Iron oxide nanoparticles (IONPs) may originate from equipment used in the manufacturing of TPs or from needles during injection. In this study, the impact of IONPs on oxidation of a model protein, rat growth hormone (rGH), was investigated under chemical stress. Hydrogen peroxide (H2O2)- and 2,2'-azobis (2-methylpropionamidine) dihydrochloride oxidized methionine residues of rGH, but unexpectedly, oxidation was suppressed in the presence of IONPs compared to a phosphate buffer control. Fourier transform infrared spectroscopy indicated splitting of the α-helical absorbance band in the presence of IONPs, whereas circular dichroism spectra showed a reduced α-helical contribution with increasing temperature for both rGH and rGH-IONP mixtures. The results collectively indicate that IONPs can increase the chemical stability of rGH by altering the kinetics and preference of amino acid residues that are oxidized, although the changes in protein secondary structure by IONPs may lead to alterations of physical stability.

Cell-penetrating peptide together with PEG-modified mesostructured silica nanoparticles promotes mucous permeation and oral delivery of therapeutic proteins and peptides.

Poor permeation across intestinal mucous barriers often limits the oral delivery of prospective therapeutic proteins and peptides (TPPs). In order to address this issue, cell penetrating peptide (CPP) together with PEG modified and pore-enlarged mesostructured silica nanoparticle (NP) were constructed to form the mucus-penetrating electrostatic particle-complexes, CPP/TPP/NP. Alone, CPP and TPP often present with poor stability, and their traditional electrostatic complex shows reduced pharmacodynamics. To provide satisfactory protection, silica NPs were loaded with CPP and TPP (CPP@NP and TPP@NP), respectively, and then CPP@NP and TPP@NP could together form CPP/TPP/NP via electrostatic interaction. As a result, CPP involvement with PEG modification showed an 8.45-, 1.62- and 5.09-fold increase in cellular uptake, exocytosis and final transcellular permeation in mucous conditions, respectively. It was found that CPP involvement mainly affected transport and exocytosis, and the PEG polymer significantly influenced mucous penetration and cellular uptake, which could further promote CPP ability for uptake and exocytosis. Additionally, NP-mediated CPP/TPP/NP showed a similar uptake mechanism with supporting carriers (clathrin-mediated endocytosis), and could strengthen transcellular routes (the endoplasmic reticulum-Golgi apparatus pathway and the lysosome route). Utilizing recombinant growth hormone (RGH) as a model TPP, oral administration of the RGH-loaded CPP/TPP/LMSN-PEG10k with hydrophilic and electroneutral properties induced 5.41- and 4.91-fold increases in pharmacodynamics in vitro and in vivo, respectively. Thus, CPP/TPP/NP significantly promoted mucous permeation and shows promising potential for oral delivery of TPPs.

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations.

The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the "alanine" alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔGtotal = -140.62 kcal mol-1), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔGtotal = -124.24 kcal mol-1). Here we report the dynamic behavior of change of "alanine" in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.

AggScore: Prediction of aggregation-prone regions in proteins based on the distribution of surface patches.

Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed "AggScore" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.

Tracing Recombinant Bovine Somatotropin Ab(Use) Through Gene Expression in Blood, Hair Follicles, and Milk Somatic Cells: A Matrix Comparison.

The use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden in the European Union. Due to the very low circulating concentration of rbST in treated animals, its direct detection is still a challenge. Therefore, the use of indirect methods to detect the ab(use) of rbST in dairy cattle appears as a good alternative. In the past few years, gene expression demonstrated its utility in screening the use of illicit substances in both humans and animals. In this study, a comparison of three types of matrices (milk somatic cells, blood, and hair follicles) was carried out to evaluate their potential use for routine control of rbST using 15 gene-expression profiles. A total of six rbST-treated cows and three control cows were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group. Samples of the three matrices were collected before rbST administration, and at three and nine days after treatment. The quality of RNA extracted was higher in the blood and hair-follicle samples than in the milk somatic cells. In the three matrices, there were significant differences in the expression of some genes, with milk somatic cells and blood presenting the the best matrices. On this note, the cyclin D1 (CCND1), interleukin 1 beta (IL-1ß), tumor necrosis factor (TNF), and insulin-like growth factor 1 receptor (IGF-1R) genes showed potential as biomarkers of rbST treatment. Therefore, blood, somatic cells, and follicle hair should be considered as promising sources of RNA, and can be used in gene-expression assays to routinely control the illicit use of rbST.

Water entrapment and structure ordering as protection mechanisms for protein structural preservation.

In this paper, molecular dynamics is used to further gain insight into the mechanisms by which typical pharmaceutical excipients preserve the protein structure. More specifically, the water entrapment scenario will be analyzed, which states that excipients form a cage around the protein, entrapping and slowing water molecules. Human growth hormone will be used as a model protein, but the results obtained are generally applicable. We will show that water entrapment, as well as the other mechanisms of protein stabilization in the dried state proposed so far, may be related to the formation of a dense hydrogen bonding network between excipient molecules. We will also present a simple phenomenological model capable of explaining the behavior and stabilizing effect provided by typical cryo- and lyo-protectants. This model uses, as input data, molecular properties which can be easily evaluated. We will finally show that the model predictions compare fairly well with experimental data.

Stability of Proteins in Carbohydrates and Other Additives during Freezing: The Human Growth Hormone as a Case Study.

Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.

Lambing rate and prolificacy in inseminated hair sheep treated with bovine somatotropin.

This study evaluated whether the administration of 50 and 100 mg bovine somatotropin (bST) at the start of estrous synchronization and at the time of artificial insemination improves lambing rate and prolificacy in hair sheep. Four hundred eighty adult hair ewes (Pelibuey, Blackbelly, Dorper, Katahdin, and their crosses) were synchronized with intravaginal sponge containing 40 mg of fluorogestone acetate. On the day of sponge insertion, ewes were assigned to three treatments: the bST-100 treatment (n = 156) received 100 mg bST at the start of synchronization (d 0) and at the time of insemination (d 14), the bST-50 treatment (n = 159) received 50 mg bST in the same schedule as the previous group, and the control (n = 165) did not receive any bST. Lambing rate and percentage of multiple births were analyzed using the GENMOD procedure of SAS. Prolificacy data were analyzed using the MIXED procedure of SAS. The IGF-1 and insulin concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the lambing rate (P = 0.06). The proportion of ewes with multiple births (P = 0.01) and prolificacy (P = 0.04) were higher in the bST-50 (54.3% and 1.57 ± 0.1) than the bST-100 (18.2% and 1.25 ± 0.1) and control (33.3% and 1.28 ± 0.1) groups. The IGF-1 and insulin concentrations were higher (P < 0.05) in the bST-treated groups, but the insulin concentration was higher (P = 0.001) in the bST-100 group than in the bST-50 group. The administration of 50 or 100 mg bST at the start of synchronization and at the time of artificial insemination does not increase lambing rate. However, the dose of 50 mg increased the proportion of multiple births and prolificacy.

[Fish growth-hormone genes: functionality evidence of paralogous genes in Levanidov's charr].

In the genome of most vertebrates growth-hormone gene is presented in a single copy, while in salmonids after one of the duplication events many genes were multiplied, including growth hormone gene. In salmonids, the growth-hormone gene exists as two independently inherited functional paralogues, gh1 and gh2. In this study, we performed a comparative analysis of gh1 and gh2 growth-hormone genes and their adjacent sequences in Levanidov's charr Salvelinus levanidovi to determine their functionality and define the potential differences. We found that both genes have the same gene structure and are composed of six exons (I-VI) and five introns (A, B, C, D, E). However, the respective gene sequences differ in length. A comparison of exons showed that the size of each exon is identical in both paralogues. The overall length of genes differs due to the varying lengths of introns. Coding sequence of both genes contains an open reading frame for 210 amino acids. We identified regulatory elements in the promoter region of both genes: TATA box, A/T-rich regions that contain binding sites for pituitary-specific transcriptional activator Pit-1, and regions responsible for interaction with other transcriptional activators and initiators, in particular hormone receptors. The obtained data indicate that both genes are functional.

Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry.

Protein therapeutics are rapidly transforming the pharmaceutical industry. Unlike for small molecule therapeutics, current technologies are challenged to provide the rapid, high-resolution analyses of protein higher order structures needed to ensure drug efficacy and safety. Consequently, significant attention has turned to developing new methods that can quickly, accurately, and reproducibly characterize the three-dimensional structure of protein therapeutics. In this work, we describe a method that uses diethylpyrocarbonate (DEPC) labeling and mass spectrometry to detect three-dimensional structural changes in therapeutic proteins that have been exposed to degrading conditions. Using ß2-microglobulin, immunoglobulin G1, and human growth hormone as model systems, we demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations.

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.

This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 µM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.

Effects of age and reproductive experience on the distribution of prolactin and growth hormone secreting cells in the anterior pituitary of a passerine.

Plasma prolactin (PRL) is released from lactotrophs in the anterior pituitary. As plasma PRL levels rise during incubation in domestic fowl, the number of lactotrophs (PRL-immunoreactive, PRL-IR cells) increases while the number of growth hormone secreting cells, somatotrophs (GH-IR cells), declines. We measured plasma PRL levels using radioimmunoassay (RIA) and examined the distribution of lactotrophs and somatotrophs in the anterior pituitary of breeding and nonbreeding zebra finches of known ages with and without prior breeding experience using fluorescent immunohistochemistry (IHC). Plasma PRL levels were higher in breeding than in nonbreeding birds, regardless of age, sex, or previous breeding history. PRL-IR cells were localized primarily, but not exclusively, to the cephalic aspect of the anterior pituitary (AP) and along the ventral margin. Birds with prior reproductive experience had more PRL-IR cells than birds with no prior reproductive experience and breeders had slightly higher PRL-IR cell counts than did nonbreeders, but there was no correlation between the number of PRL-IR cells and plasma PRL levels. GH-IR cells were concentrated in the caudal aspect of the AP with some cells in the cephalic lobe, but numbers did not differ between any of the groups studied. An increase in PRL-IR cells corresponded with an increase in GH-IR cells. An increase in lactotroph number with reproductive experience in zebra finches may facilitate future reproductive events by allowing for more robust PRL secretion and increased reproductive success.

Influence of L-homoarginine as an analogue of L-arginine on the heat-induced aggregation of proteins.

The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.

Control of rhGH Release Profile from PEG-PAF Thermogel.

Poly(ethylene glycol)-poly(l-alanine-co-l-phenyl alanine) diblock copolymers (PEG-PAF) of 2000-990 Da (P2K) and 5000-2530 Da (P5K) with the different molecular weights of PEGs, but having a similar molecular weight ratio of hydrophobic block to hydrophilic block were synthesized to compare their solution behavior and corresponding protein drug release profiles from their in situ formed thermogels. The PEG-PAF aqueous solutions underwent heat-induced sol-to-gel transition in a concentration range of 18.0-24.0 wt % and 8.0-12.0 wt % for P2K and P5K, respectively. P5K formed bigger micelles than P2K, of a broad distribution, whereas the PAF blocks of P5K developed richer in α-helix than those of P2K in the core of the micelles. As the temperature increased, the micelles underwent dehydration of the PEG, which led to the aggregation of micelles, while the secondary structure of PAF was slightly affected during the sol-to-gel transition. The P5K exhibited higher tendency to aggregate and formed a tighter gel than P2K. Upon injection into the subcutaneous layer of rats, both polymer aqueous solutions formed a biocompatible gel with typical mild inflammatory tissue responses. Recombinant human growth hormone (rhGH) maintained its stability without forming any aggregates in both sol (4 °C) and gel (37 °C) states of the PEG-PAFs. Even though P2K and P5K have a similar molecular weight ratio of hydrophobic block to hydrophilic block, the P5K system exhibited a reduced initial burst release, improved bioavailability, and prolonged therapeutic duration of the rhGH, compared to the P2K system. The current research suggests that a drug release profile is a complex function of self-assembling carriers and incorporated drugs, and thus, a promising protein delivery system could be designed by adjusting the molecular parameters of a thermogel.