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1.
Protein Sci ; 30(8): 1686-1700, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34060159

RESUMO

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the CH 2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.


Assuntos
Anticorpos Monoclonais , Estabilidade Proteica , Eletricidade Estática , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Varredura Diferencial de Calorimetria , Fluorometria , Espectrometria de Massa com Troca Hidrogênio-Deutério , Nanotecnologia , Dobramento de Proteína , Desdobramento de Proteína , Cloreto de Sódio/química , Termodinâmica
2.
Trends Biotechnol ; 39(3): 298-310, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32807530

RESUMO

Intracellular delivery of therapeutic antibodies is highly desirable but remains a challenge for biomedical research and the pharmaceutical industry. Approximately two-thirds of disease-associated targets are found inside the cell. Difficulty blocking these targets with available drugs creates a need for technology to deliver highly specific therapeutic antibodies intracellularly. Historically, antibodies have not been believed to traverse the cell membrane and neutralize intracellular targets. Emerging evidence has revealed that anti-DNA autoantibodies found in systemic lupus erythematosus (SLE) patients can penetrate inside the cell. Harnessing this technology has the potential to accelerate the development of drugs against intracellular targets. Here, we dissect the mechanisms of the intracellular localization of SLE antibodies and discuss how to apply these insights to engineer successful cell-penetrating antibody drugs.


Assuntos
Anticorpos Antinucleares , Produtos Biológicos , Sistemas de Liberação de Medicamentos , Lúpus Eritematoso Sistêmico , Anticorpos Antinucleares/administração & dosagem , Anticorpos Antinucleares/química , Anticorpos Antinucleares/metabolismo , Produtos Biológicos/administração & dosagem , Humanos , Espaço Intracelular , Lúpus Eritematoso Sistêmico/tratamento farmacológico
3.
Cytokine ; 65(2): 167-74, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24345576

RESUMO

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1=isoform 2

Assuntos
Dissulfetos/metabolismo , Interleucina-17/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Interleucina-17/química , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Redobramento de Proteína , Soluções , Espectrometria de Fluorescência
4.
Artigo em Inglês | MEDLINE | ID: mdl-22771237

RESUMO

This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125 psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200-300 proteins (Seppro SuperMix). The autosampler was dual-purposed for both injection and fraction collection. Both the flow-through and SuperMix bound proteins were collected in an automated fashion. Three samples could be depleted consecutively before the system required user intervention, and up to nine samples could be depleted within a 24 h period. This study documents the validation of the instrument performance with a 90-patient sample set, demonstrating overall CVs for 86 of the 90 samples to be within the 95% confidence intervals. Additionally, there was excellent reproducibility within the same patient (biological replicates) across days.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Área Sob a Curva , Cromatografia de Afinidade/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Humanos , Reprodutibilidade dos Testes
6.
J Mol Recognit ; 25(3): 114-24, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22407975

RESUMO

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.


Assuntos
Anticorpos Imobilizados/química , Anticorpos Monoclonais/química , Simulação por Computador , Mapeamento de Epitopos , Modelos Moleculares , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos , Citocromos c/química , Citocromos c/imunologia , Medição da Troca de Deutério , Humanos , Ligação de Hidrogênio , Interleucina-13/química , Interleucina-13/imunologia , Interleucina-17/química , Interleucina-17/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ligação Proteica , Estrutura Quaternária de Proteína , Homologia Estrutural de Proteína , Propriedades de Superfície
7.
J Mol Recognit ; 25(3): 174-83, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22407981

RESUMO

There are a number of proteins whose active forms are non-covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild-type (wt) proteins. We have used tumor necrosis factor-α (TNF-α) to establish that it is possible to use mass spectrometry to monitor the non-covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5 Da errors mass accuracy of theoretical mass at 50 kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF-α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti-TNF-α antibody. Dissociation and re-association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non-covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non-covalent drug complexes for the purpose of altering biological activity.


Assuntos
Desnaturação Proteica , Espectrometria de Massas por Ionização por Electrospray , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Dimetil Sulfóxido/química , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica , Redobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Solventes/química , Fator de Necrose Tumoral alfa/genética
8.
J Mass Spectrom ; 46(7): 677-88, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21744417

RESUMO

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.


Assuntos
Dissulfetos/química , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Espectrometria de Massas em Tandem
9.
Cytokine ; 53(1): 107-14, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20674388

RESUMO

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.


Assuntos
Interleucina-17/química , Interleucina-17/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Redobramento de Proteína , Dicroísmo Circular , Escherichia coli/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Interleucina-17/isolamento & purificação , Espectrometria de Massas , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
10.
Biopolymers ; 94(3): 350-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20091676

RESUMO

The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP-1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester-peptide segment was synthesized using the sulfonamide safety-catch linker and 9-fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full-length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP-1 cells, as expected. CCL2 was crystallized and the structure was determined by X-ray diffraction at 1.9-A resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9-Thr10-Cys11, which form an intersubunit antiparallel beta-sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety-catch linker and NCL.


Assuntos
Quimiocina CCL2/química , Quimiocina CCL2/síntese química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Quimiocina CCL2/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Dobramento de Proteína , Multimerização Proteica , Ensaio Radioligante
11.
Anal Chem ; 81(21): 8715-23, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19788250

RESUMO

Three different analysis platforms using LC-MS were successfully developed for pharmacokinetic (PK) studies of an antibody drug in serum. These analysis platforms can be selectively used for different types of protein drugs, which ranged from a very specific for a particular drug (antibody enrichment) to a less specific for any antibody drugs with an Fc domain (protein A enrichment), and to a very generic method that can be used for any protein drugs (albumin depletion method). In this manner, the three platforms will be applicable to a wide range of antibody therapeutic studies for different species. The analysis using an albumin depletion method (with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) achieved the detection of the drug (to 1 ng) in an aliquot of serum (30 microL) with a 5-order magnitude of linearity. The analysis using protein A enrichment (with SDS-PAGE) achieved the detection of the drug at a 50-fold lower level (to 0.02 ng). Without the use of SDS-PAGE for separation, the use of protein A enrichment achieved the detection to 10 ng and using the antidrug antibody enrichment achieved the detection to 0.1 ng, with a similar linear dynamic range. These three analysis platforms produced good agreement with a mimic PK study of the drug in monkey serum, as compared to the enzyme-linked immunosorbent assay (ELISA) approach. In addition, these analysis platforms can be selectively applied for PK studies of drugs with different requirements of development time and resources. Such as, the antibody enrichment method can be used in a high-throughput manner but limited to a specific protein drug only. On the other hand, the albumin depletion method can be applied to many types of protein drugs but with the laborious sample preparation steps (SDS-PAGE and the subsequent in-gel digestion). When antidrug antibodies are not available for antibody drugs or the sensitivity requirement is not stringent (e.g., >10 ng), using protein A enrichment (without using SDS-PAGE) seems to be a good choice for PK studies which require fast throughput.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Macaca fascicularis/metabolismo , Proteínas Recombinantes de Fusão/sangue , Albumina Sérica/química , Proteína Estafilocócica A/metabolismo
13.
Biotechnol Bioeng ; 103(1): 162-76, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19137588

RESUMO

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O-linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N-terminal clipping had increased level of O-linked glycosylation in the linker region, giving credence to the hypothesis that O-linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N-terminal clipping had longer in vivo serum half-life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules.


Assuntos
Biotecnologia/métodos , Proteínas Recombinantes de Fusão/biossíntese , Animais , Linhagem Celular , Cricetinae , Humanos , Camundongos
14.
Mol Immunol ; 45(7): 1837-46, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18157932

RESUMO

A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab')(2), and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab')(2). Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab')(2) or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab')(2)s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab')(2) and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.


Assuntos
Artrite Reumatoide/imunologia , Bactérias/enzimologia , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Processamento de Proteína Pós-Traducional , Líquido Sinovial/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/isolamento & purificação , Imunoglobulina G/química , Dados de Sequência Molecular , Peptídeos/imunologia , Coelhos
15.
J Pept Sci ; 12(1): 25-32, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15942931

RESUMO

Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.


Assuntos
Quimiocina CCL2/síntese química , Quimiocina CCL2/farmacologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Ligação Competitiva , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL2/química , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Humanos , Ligantes , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Receptores CCR2 , Receptores de Quimiocinas/efeitos dos fármacos , Relação Estrutura-Atividade
16.
J Pept Sci ; 12(5): 354-60, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16285024

RESUMO

Human monocyte chemoattractant protein 1 (MCP-1, CCL2) is a 8.6-kDa protein that has been implicated in a number of diseases including atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease and cancer. As part of a program to identify antibodies against MCP-1, we synthesized site-specific, biotinylated human MCP-1 analogs to be used for panning of an antibody phage display library. In contrast to material obtained from random biotinylation, the site-specific biotinylated analogs were homogeneous and retained full activity.


Assuntos
Quimiocina CCL2/química , Sequência de Aminoácidos , Biotinilação , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL2/síntese química , Quimiocina CCL2/farmacologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Nutr Biochem ; 16(5): 272-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15866226

RESUMO

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their implications for protein bioactivity and infant nutrition need to be considered.


Assuntos
Lactalbumina/genética , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Sequência de Bases , Feminino , Genótipo , Heterozigoto , Humanos , Proteínas do Leite/genética , Dados de Sequência Molecular , Filipinas
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