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High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli (1996)

Jansson, Magnus ; Li, Yu-Chin ; Jendeberg, Lena ; [et al.]

Springer

Journal of biomolecular NMR 7 (1996), S. 131-141

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ISSN: 1573-5001

Keywords: Biosynthetic enrichment ; Insulin-like growth factor ; Staphylococcal protein A ; Bovine panceatic trypsin inhibitor ; Triple-resonance NMR

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Summary An approach to produce 13C-and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40–200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C6-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C6-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purified IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20–40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00203823

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Kinetic analysis of the interaction between protein a domain variants and human Fc using plasmon resonance detection (1995)

Jendeberg, Lena ; Persson, Björn ; Andersson, Roland ; [et al.]

Chicester [u.a.] : Wiley-Blackwell

Journal of Molecular Recognition 8 (1995), S. 270-278

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ISSN: 0952-3499

Keywords: association rate affinity ; biospecific interaction analysis ; dissociation rate ; Kinetics ; protein engineering ; staphylococcal protein A ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Medicine

Notes: A real time biospecific interaction analysis (BIA) was performed to study the specific interaction between the Fc portion of human immunoglobulin G1 (Fc1) and a one domain analogue (designated Z) of staphylococcal protein A, in monovalent (Z) and divalent (ZZ) forms, and five different single amino acid substituted Z variants (L17D, N28A, F30A, I31A, K35A). Experimental BIA data were used to calculate association rate constants (Kon), dissociation rate constants (koff). The divalent form (ZZ) showed a higher affinity for Fc1 mainly because of a slower off rate. Out of the five mutant Z protein, four (L17D, N28A, I31A, K35A) four had the major effect to Fc1 compared to the parent Z molecule. Surprisingly, two (L17D, I31A) of these four had the major effect of a decreased binding energy as a lowered kon while the other two (N28A, K35A) mutant proteins showed an increased koff as the major kinetic difference from Z in their binding to Fc1. For five of the six different Z variants, as well as for the ZZ molecule, calculated kaff and calculated differences in binding free energies relative to the parent Z molecule (ΔΔG), are in good agreement with the corresponding values obtained in a competitive displacement assay using radioactively labeled Z as a tracer (Cedergren et al., (1993) Prot. Eng. 6, 441-448). However, the I31A variant, with a measured kon that was more than three orders of magnitude lower than that of Z in the BIA assay, showed a significant weaker affinity to FcI when calculated from BIA data compared ot the competitive displacement assay. The discrepancy between these two methods for Z(I31A) is discussed as well as possible explanations for the unexpected large effect of lowered kon for two of the mutant Z proteins.

Additional Material: 6 Ill.