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1.
Recombinant human growth hormone (rhGH) is used for the treatment of several disorders. Structural integrity of rhGH is of critical importance for its clinical use and modifications thereof may act as markers in situations such as rhGH doping, as illegal rhGH-abuse in sports is of increasing interest. In the current study we investigated homogeneity of Norditropin, a recombinant human growth hormone frequently used in medicine, expressed in E. coli, strain MC1061. The most recent proteomics technologies including 2-DE, MALDI-MS followed by MALDI-MS/MS and LC-MS followed by LC-MS/MS were used for the characterisation of rhGH. MALDI-TOF-TOF and electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22 126.0 Da and some additional minor components. Electrospray LC-MS/MS of the enzymatically digested Norditropin sample showed deamidation of N(12)N(149) and N(159), oxidation of M(14), M(125) and M(170) and one amino acid exchange V(14) for M(14) present in <1% of Norditropin. While deamidation and oxidation may be due to technical reasons, the single amino acid exchange may reflect infidelity of translation rather than codon usage and copy editing by E. coli. 相似文献
2.
Background
The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin?), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides. 相似文献3.
Mass spectrometric analysis of innovator,counterfeit, and follow‐on recombinant human growth hormone
Haitao Jiang Shiaw‐Lin Wu Barry L. Karger William S. Hancock 《Biotechnology progress》2009,25(1):207-218
We have performed a detailed characterization of recombinant human growth hormone that included the identification of the entire sequence with disulfide linkages as well as subtle modifications by a sensitive liquid chromatography coupled online with tandem mass spectrometry (LC‐MS) approach using the accurate peptide mass (FTICR MS) and sequence assignment (MS/MS measurement). The extent of oxidation, deamidation, and chain cleavages were measured by the ratio of peak areas of the nonmodified peptide vs. the sum of peak area of the nonmodified and modified peptides in the same LC‐MS analysis. The subtle but distinct differences were found in the recombinant human growth from the three manufacturers (the follow‐on, counterfeit, and the original innovator products). In relative comparison, the follow‐on product had the highest degree of oxidation at methionine residues, followed by the counterfeit product, and the original innovator product had the least amount of oxidation at all three sites with the similar oxidation order. In cases, the oxidation order was Met14 > Met125 > Met170. In contrast, the follow‐on had the least amount of deamidation at aspargine (Asn149), and the counterfeit had the highest degree of deamidation at this site. For the chain cleavage, the follow‐on product had the highest cleavage occurring at T 10 peptide (between Asn99 and Ser100), the counterfeit had the highest cleavage on T4 peptide, (between Glu30 and Phe31), and the original innovator product with the least amount of cleavages on both sites. These subtle but distinct differences are likely because of nonidentical manufacturing, formulation procedures, and storage conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
4.
There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quantities of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E. coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 degrees C. Determination of the yield of recombinant hPL by SDS-PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8M urea and purified by a His6 tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL. 相似文献
5.
人生长激素突变体的制备及其对受体亲和力和生物活性的研究 总被引:1,自引:0,他引:1
本文采用DNA重组技术制备了三种重组人生长激素(recombinant human growthhormone,rhGH)突变体,并测定了其受体亲和力及对去垂体大鼠的促体重增加活力。实验结果显示其受体亲和力大小依次为:rhGH-E174A>rhGH-G120R>rhGH>rhGH-G120T。 rhGH-G120R和rhGH-G120T二种突变体相比rhGH来说,对去垂体大鼠促体重增加生物活性明显降低;rhGH-E174A突变体则完全丧失生物活性。由于rhGH-E174A的受体亲和办高于rhGH,表明它是一种较强的hGH拮抗剂,可能成为一种药物,在临床上用于治疗因hGH分泌过多而引起的疾病(例如巨人症,肢端肥大症等)。 相似文献
6.
Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data 总被引:2,自引:0,他引:2
Johansson C Samskog J Sundström L Wadensten H Björkesten L Flensburg J 《Proteomics》2006,6(16):4475-4485
The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels. 相似文献
7.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1996,1292(1):168-176
Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively. 相似文献
8.
A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry. 总被引:3,自引:2,他引:1
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P. T. Jedrzejewski A. Girod A. Tholey N. Knig S. Thullner V. Kinzel D. Bossemeyer 《Protein science : a publication of the Protein Society》1998,7(2):457-469
The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization. 相似文献
9.
Apidaecins refer to a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. Accumulating evidence that proline-rich antimicrobial peptides are not-toxic to human and animal cells makes them potential candidates for the development of novel antibiotic drugs. However, the mechanism of action was not fully understood. In this study, antibacterial mechanism of apidaecins was investigated. iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered cytoplasmic proteins of Escherichia coli incubated with one isoform of apidaecins--apidaecin IB. The production of the chaperonin GroEL and its cofactor GroES, which together form the only essential chaperone system in E. coli cytoplasm under all growth conditions, was decreased in cells incubated with apidaecin IB. The decreasing of the GroEL-GroES chaperone team was further found to be involved in a new antibacterial mechanism of apidaecins. Our findings therefore provide important new insights into the antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides. 相似文献
10.
Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone 总被引:4,自引:0,他引:4
B A Johnson J M Shirokawa W S Hancock M W Spellman L J Basa D W Aswad 《The Journal of biological chemistry》1989,264(24):14262-14271
In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification. 相似文献
11.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2018,1866(7):767-774
Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit–subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens. 相似文献
12.
van der Hel OL van der Luijt RB Bueno de Mesquita HB van Noord PA Slothouber B Roest M van der Schouw YT Grobbee DE Pearson PL Peeters PH 《Analytical biochemistry》2002,300(2):206-211
Metal-catalyzed oxidation was used to identify metal-binding His residues in bovine growth hormone (bGH), which has not been characterized well crystallographically due to a high propensity of bGH to aggregate. bGH was exposed to Cu(2+) and ascorbate (ascorbate/Cu(2+)/O(2)). 2-Oxo-His formation was identified by HPLC-tandem mass spectrometry (MS/MS) analysis of a tryptic digest. Two 2-oxo-His-containing fragments were detected, T2(O) (MH(2+)(2) = 748.8) and T20(O) (MH(+) = 528.3), both masses corresponding to the addition of only one oxygen atom (+16 amu) to the respective native fragments, T2 and T20. T2 contains (20)His and (22)His, and T20 contains (170)His. Quantitative HPLC-MS/MS analysis shows the following order of reactivity: (170)His > (22)His > (20)His. Solvent-accessible surface area calculations determined (22)His and (170)His to be 26 and 35% solvent exposed, respectively, while (20)His is 65% solvent exposed. The presence of an analogous metal-binding site in human growth hormone, which is located in the hydrophobic core, and our experimental finding that oxidation was greatest for (22)His and (170)His in bGH suggests that (22)His and (170)His of bGH participate in metal binding. This result is supported by a previously predicted tertiary structure of bGH and compared with the location of metal-binding His residues of human growth hormone. 相似文献
13.
The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week. 相似文献
14.
Ge Y Lawhorn BG ElNaggar M Sze SK Begley TP McLafferty FW 《Protein science : a publication of the Protein Society》2003,12(10):2320-2326
Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 +/- 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R(28)-V(31), E(95)-F(107), and K(216) of 22%, 28%, and 34%, respectively, but with a possible (approximately 4%) fourth site at V(125)-D(150). However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable approximately 13% oxygen at the V(125)-D(150) site. For the "bottom-up" approach, detection of the approximately 4% oxidation at the V(125)-D(150) site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just approximately 4% of this, but the uniqueness of its mass value (approximately 1-2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V(125)-D(150) until K(216) is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K(216), H(137), and D(139) are conserved residues. 相似文献
15.
The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41-49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism. 相似文献
16.
Although the effect of sucrose on the physical stability of proteins has been well documented, its impact on their chemical stability is largely unknown. The aim of this study was to investigate the potential effects of sucrose on the structural conformation of human brain natriuretic peptide [hBNP (1-32)] and the synthetic human parathyroid hormone [hPTH (1-34)], and link these effects to chemical degradation pathways of these peptides. The stability of hBNP (1-32) and hPTH (1-34) was studied at pH 5.5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas oxidation and deamidation products were measured by reversed phase (RP) HPLC. Fourier transform infrared (FT-IR) spectroscopy was used to study the peptides' conformation. Sucrose retarded aggregation, deamidation, and oxidation of hBNP (1-32) and hPTH (1-34), with a maximum effect at relatively high concentrations (as much as 1 m). FT-IR spectroscopy indicated that sucrose maintained the native conformation of hBNP (1-32) and induced small conformation changes in the hPTH (1-34) structure. Sucrose enhanced the stability of hBNP (1-32) and hPTH (1-34) in liquid formulations. The stabilizing effect of sucrose was due to a large extent to retardation of oxidation and deamidation of hBNP (1-32) and hPTH (1-34). 相似文献
17.
Total protein variation (up to ninety-five different positions) was revealed by two-dimensional electrophoresis (2-DE) in 18 isolates from populations of M. arenaria (6 isolates), M. incognita (10), M. javanica (1) plus an unclassified isolate in a previously reported study. Isolates of M. arenaria, M. javanica, Meloidogyne sp., and M. incognita formed two separate groups defined on the basis of two sets of protein positions that could be considered as diagnostic characters, but we could not identify these proteins by MALDI-TOF. To identify these marker positions, nano-liquid chromatography as peptides separation method was coupled to an ion-trap mass spectrometer for induced real-time fragmentation of eluted peptides. Group diagnostic proteins for M. incognita and M. arenaria were in-gel digested and on line analyzed by tandem mass spectrometry (LC-MS/MS). Six proteins out of seven selected spots were unambiguously identified by the analysis of the corresponding MS/MS (MS2) spectrum from parent ions fragmentation: Actin, Enolase, CG3752-PA protein similar to Aldehyde Dehydrogenase, HSP-60 and Translation initiation factor elF-4A. In M. incognita sample, de novo sequencing experiment of doubly charged ion at m/z=936.9 Da in spot 29 identified as enolase, reveals three residue substitutions (K to T, N to T, and D to E) when tentative sequence was compared with that of Anisakis simplex and Onchocerca volvulus enolase, thus three SNPs (single nucleotide polymorphisms) were also possibly identified. 相似文献
18.
Jim W. Xuan Dongmei Wu Yuzhen Guo Seema Garde Madhulika Baijal-Gupta Joseph L. Chin 《Journal of cellular biochemistry》1997,65(2):172-185
PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc. 相似文献
19.
Catai JR Sastre Toraño J Jongen PM de Jong GJ Somsen GW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,852(1-2):160-166
The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products. 相似文献
20.
The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone. 相似文献