Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity☆

Agarwal, Saurabh; Jha, Shweta; Sanyal, Indraneel; Amla, D. V.

doi:10.1016/j.jbiotec.2010.03.008

Cited by 15 publications

(8 citation statements)

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“…In fact, loss of the negative charge displayed at the P13 position and substitution to a positive Lys residue clearly enhanced the rate of loop insertion and correlated to both an increase in polymerization rate and a slight decrease in thermal stability. The latter two effects have been observed for various substitutions of the charged E342 residue (P17) as well; however, this has been linked to a dramatic decrease in inhibitory activity of more than 40% 69,76 and thus differs from the effect of the E346 residue discussed here. The importance of the P13 residue in regulating the rate of loop insertion has to our knowledge only been investigated for PAI-1 that has a Val at this Page 34 of 45 Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 position.…”

Section: Acs Paragon Plus Environmentmentioning

confidence: 58%

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

et al. 2017

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Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease-serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subsequent insertion into central β-sheet A, and covalent trapping of the target protease. In this study, we present the first detailed accelerated molecular dynamics simulation of the insertion of the fully cleaved RCL in α-1-antitrypsin (αAT), the archetypal member of the family of human serpins. Our results reveal internal water pathways that allow the initial incorporation of side chains of RCL residues into the protein interior. We observed structural plasticity of the helix F (hF) element that blocks the RCL path in the native state, which is in excellent agreement with previous experimental reports. Furthermore, the simulation suggested a novel role of hF and the connected turn (thFs3A) as chaperones that support the insertion process by reducing the conformational space available to the RCL. Transient electrostatic interactions of RCL residues potentially fine-tune the serpin inhibitory activity. On the basis of our simulation, we generated the αAT mutants K168E, E346K, and K168E/E346K and analyzed their inhibitory activity along with their intrinsic stability and heat-induced polymerization. Remarkably, the E346K mutation exhibited enhanced inhibitory activity along with an increased rate of premature structural collapse (polymerization), suggesting a significant role of E346 in the gatekeeping of the strain in the metastable native state.

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Section: Acs Paragon Plus Environmentmentioning

confidence: 58%

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

et al. 2017

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“…The M351E mutation has also been used to increase expression in insect cells, with the additional modification of an N‐terminal fusion protein to facilitate α 1 ‐AT secretion . The addition of a signal peptide or fusion protein is a common technique used to increase recombinant protein expression, and has been used to improve α 1 ‐AT yields from E. coli , yeast, and plants . In the first study to overexpress α 1 ‐AT using cultured mammalian cells, modification to the 5′ untranslated region and endogenous signal peptide improved expression up to fivefold, however, more recent studies using the wild type endogenous signal peptide achieved superior yields .…”

Section: Rationale For α1‐at Mutagenesis and Engineering Of Novel Promentioning

confidence: 99%

Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques

Scott

Sheffield

2019

Protein Science

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α1‐Antitrypsin (α1‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α1‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α1‐AT, specific mutants of interest, and providing an appended catalog of the >200 α1‐AT mutants published to date.

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“… a Defined medium for fermentation b Optimized strain and growth condition from [13] c Current Study NR not reported …”

Section: Figmentioning

confidence: 99%

Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli

Krishnan

Hedstrom

Hebert

et al. 2017

Methods in Molecular Biology

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Well-established genetic manipulation procedures along with a fast doubling time, the ability to grow in inexpensive media, and easy scaleup make Escherichia coli (E. coli) a preferred recombinant protein expression platform. Human alpha-1 antitrypsin (AAT) and other serpins are easily expressed in E. coli despite their metastability and complicated topology. Serpins can be produced as soluble proteins or aggregates in inclusion bodies, and both forms can be purified to homogeneity. In this chapter, we describe an ion-exchange chromatography-based protocol that we have developed involving the use of two anion-exchange columns to purify untagged human AAT from E. coli. We also outline methods that can be used to determine the inhibitory activity of both AAT in cell lysates and purified AAT. Our protocol for the purification of bacterially expressed AAT yields pure and active protein at 6–7 mg/l culture.

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Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity☆

Cited by 15 publications

References 36 publications

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques

Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli

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Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity☆

Cited by 15 publications

References 36 publications

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli

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Product

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Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques